PT Cohen2001 PDF

Cisplatin: From DNA Damage
to Cancer Chemotherapy
SETH M. COHEN AND
S T E P H E N J. L I P P A R D 1
Department of Chemistmj
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
I. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
II. Kinetic and Thermodynamic Aspects of Protein Binding
to Cisplatin-DNA Adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
A. Proteins That Bind to Cisplatin-Modified D N A . . . . . . . . . . . . . . . . . . . . 98
B. H M G - D o m a i n Protein Binding to Cisplatin Adducts:
Thermodynamic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
C. H M G - D o m a i n Protein Binding to Cisplatin Adducts:
Kinetic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
III. Structure of an H M G D o m a i n - C i s p l a t i n - D N A Ternary Complex . . . . . . . . 104
A. Cisplatin-DNA Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
B. H M G i D o m A - C i s p l a t i n - D N A Structure . . . . . . . . . . . . . . . . . . . . . . . . . 107
C. Mutant H M G - D o m a i n Protein Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
IV. Other Consequences of Cisplatin Treatment of Cells . . . . . . . . . . . . . . . . . . . 112
A. Telomere Shortening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
B. TATA-Binding, H1, and AAG Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
C. Apoptosis and Ubiquitination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
V. Development of New Platinum Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . 117
A. Mononuclear Platinum Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
B. Polynuclear Platinum Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
C. Combinatorial/Parallel Synthesis Approaches . . . . . . . . . . . . . . . . . . . . . . 120
D. Screening Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
E. Steroid Hormones, Cisplatin, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . 122
VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Cisplatin [c/s-DDP, cis-diamminedichloroplatinum(II)] is a potent antieancer

drug that has been used successfully to treat tumors of the head, neck, lungs, and
genitourinary tract. The biological activity of eisplatin was discovered serendipi-
tously more than 30 years ago, and since that time research efforts have focused
on elucidating its mechanism of action. The present review provides a historical
perspective of our attempts to understand this complex phenomenon and the
results of recent work that guides our current activities in this field. Continued
1Author to w h o m correspondence should be addressed. Telephone: (617) 253-1892. Fax: (617)

258-8150. E-maih lippard@lippard.mit.edu.
Progressin NucleicAcid Research Copyright© 2001 by AcademicPress.

and MolecularBiology,Vol.67 93 All rightsof reproductionin an),formreserved.
0079-6603/01 $35.0(I
94 SETH M. COHEN AND STEPHEN J. LIPPARD
efforts to understand the mechanism of genotoxicity of cisplatin are expected

to lead to the discovery of new drugs and combinations for the improvement of
cancer chemotherapy. © 2001 AcademicPress.
Cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] is a relatively simple

inorganic compound that has had a major impact on the treatment of cancer
(Fig. 1). The biological activity of cisplatin was discovered serendipitously about
125 years after its first reported synthesis (1). Its utility in the treatment of
cancer is a paradigm for the use of metals in medicine (2). Today, cisplatin and
carboplatin (Fig. 1), an analog of cisplatin, are among the most widely prescribed
anticancer drugs, with annual sales exceeding $500 million (3).
Several recent reviews are available describing many of the chemical and
biological studies performed to elucidate the genotoxic mechanism of cisplatin.
These reports cover a broad range of subjects including the identification of
DNA as the target (4), structures of cisplatin-DNA adducts (5), interactions
of various proteins with cisplatin-modified DNA (6, 7), cellular consequences
of protein binding (8), and the development of new platinum anticancer drugs
(3). The sum of these investigations, which are thoroughly examined in these
reviews, provides the basis for several hypotheses to explain how the drug elicits
its antitumor effect.
Because these many excellent reviews have appeared recently (3-8), this ar-
ticle focuses on the most current discoveries from our laboratory. A brief back-
ground and perspective are provided, followed by several subjects, including
protein recognition of cisplatin-damaged DNA, structural studies on protein-
cisplatin-DNA ternary complexes, newly uncovered cytotoxic consequences of
cisplatin treatment, and parallel synthesis efforts toward the development of
novel platinum drugs. This range of topics both communicates the latest de-
velopments in understanding the mechanism of action of cisplatin and outlines
future prospects for cisplatin-related research.
I. Background
The potential utility of platinum compounds as anticancer agents was first
recognized following the demonstration of their ability to prevent bacterial cell
division (9). Subsequent investigations identified cisplatin as the most active
compound (10-12), and a decade of clinical trials ultimately led to FDA approval
in 1978. Cisplatin is used to treat a variety of cancers, including tumors of the
head, neck, lung, and genitourinary tract. Its greatest success is against testicular
cancer, a previously lethal disease with historically low survival rates before the
introduction of cisplatin (13).
BIOCHEMISTRY OF CISPLATIN 95
H3N CI H3N 0% "'/ CI/ ~NH3
cisplatin carboplatin trans-DDP
[Cr]extracellular
= - 100mM
CellMembrane
[Cl']intracellular = -2-30 mM Aquation
DNA H3N~ /OH2~ 2+ RNA

/Pt
H3N ~OH2
Proteins// Glutathione
FIG. 1. Stru•tures•f•is••atin(c/s-DDE•eft)••arb•••atin(•enter)•andtheinactivetransis•mer
trans-DDP (right). The general scheme for the in vivo reactivity of cisplatin is depicted. Cisplatin
and carboplatin are administered intravenouslywhere extracellular chloride levels are high. Upon
entering cells, the large drop in chloride ion concentration causes the platinum compounds to un-
dergo aquation reactions. Exchange of the anionic chloride or 1,1-cyclobutanedicarboxylateligands
for water molecules gives a platinum complex with a positive charge that migrates to polyanionic
nucleic acids. The platinum complex will react with many cellular targets, including proteins and
glutathione, but the ~1% of the platinum that reaches the nuclear DNA is responsible for the
genotoxicityof the compound.
O f interest to inorganic chemists was the observation that the trans iso-
mer, trans-diamminedichloroplatinum(II) or t r a n s - D D P (Fig. 1), has no ac-
tivity against tumors. A simple difference in stereochemistry conveys dramat-
ically different levels of activity. While the clinical potential of cisplatin was
being evaluated, our laboratory was investigating the interaction of heavy-metal
complexes with nucleic acids (14, 15 ). One objective of these experiments was to
sequence DNA in the electron microscope using a "beads-on-a-string" concept
afforded by specific derivatization of nucleotides with electron-dense heavy-
metal labels (16). During these investigations, platinum compounds were iden-
tified that bound to DNA through only covalent, covalent and intercalative, or
only intercalative modes (17). The last class of compounds included several plat-
inum terpyridine complexes (18, 19) that afforded the first metallointercalators.
The descendents of these early compounds constitute a substantial area of active
chemical research today (20).
When sufficient evidence had accumulated to identify DNA as the rel-
evant biological target of cisplatin (4), our experience in platinum-nucleic
acid chemistry prompted us to pursue the stereochemistry issue. Both cis- and
trans-DDP shorten and unwind supercoiled circular DNA (21, 22), binding
almost exclusively at the N7 nitrogen atom in purine bases (23), with a prefer-
ence for guanine over adenine (24). Several types of DNA adducts occur with
both platinum compounds, including monofunctional, intrastrand crosslinks,
and interstrand crosslinks. The active compound, c/s-DDP, forms predomi-
nantly 1,2-intrastrand d(GpG) and d(ApG) crosslinks and, to a lesser extent,
1,3-intrastrand and interstrand crosslinks. The inactive compound, trans-DDP,
forms predominantly 1,3-intrastrand and interstrand crosslinks. These discov-
eries suggested that the toxicity of cisplatin originated from the 1,2-intrastrand
crosslinks, which the geometry of trans-DDP does not allow (Fig. 2). Indeed,
subsequent work indicated that a positive response of patients to cisplatin
correlates with the number of 1,2-intrastrand crosslinks found in tissue
samples (25).
FIG. 2. Scheme of different crosslinks formed on DNA by platinum drugs. Several types of
adducts are formed, including (in order from left to right) 1,2-intrastrand crosslinks, interstrand
crosslinks, monofunctional adducts, and DNA-protein crosslinks. Cisplatin forms predominantly
1,2-intrastrand crosslinks, and these adducts are believed to be the most important lesions for the
genotoxiceffectsof the drug.
BIOCHEMISTRYOF CISPLATIN 97
Once the basis for the stereoehemical preference for cis-DDP had been
established, the focus of our work turned to investigating the structure, bio-
chemical consequences, and cellular responses of cisplatin damage to DNA. Cis-
platin 1,2-intrastrand erosslinks inhibit both replication (26, 2 7) and transcription
(28-31). Of primary importance was the discovery of structure-specific recogni-
tion protein 1 (SSRP1), a mammalian cellular factor that binds with specificity
to cisplatin-modified DNA (32, 33). This protein contains a high-mobility group
(H MG) D NA-binding motif (34). Additional studies demonstrated that a range
of HMG-domain proteins, including transcription and chromatin architectural
factors, could bind to cisplatin-damaged DNA (35-37). HMG-domain proteins
specifically recognize the 1,2-intrastrand crosslinks formed by eisplatin, but not
adduets formed by trans-DDP, supporting the hypothesis that 1,2-intrastrand
adduets were significant in the mechanism of action. The ability of HMG-domain
proteins to potentiate the action of cisplatin in vivo is supported by experiments
showing that yeast knockout mutants lacking the HMG-domain protein Ixrl,
intrastrand crosslink recognition protein 1, are more resistant to eisplatin than
are wild-type cells producing the protein (38-40). The dual discoveries that
HMG-domain proteins could bind with high affinity to eisplatin 1,2-intrastrand
crosslinks and that this recognition could elicit an observable effect in cells
treated with cisplatin were evaluated within the framework of various hypothe-
ses for how these proteins might mediate the genotoxieity of the drug (41, 42).
One possible pathway by which cisplatin might effect genotoxicity is through
"repair shielding" (38). Cisplatin adduets are removed primarily by enzymes of
the nucleotide excision repair (NER) pathway (43, 44). When HMG-domain
proteins such as HMG1 bind to eisplatin lesions, however, NER can be blocked
(43, 44), allowing the damage to persist. The resulting cisplatin-DNA adduets
block replication and transcription. Another mechanism by which cisplatin can
damage the cell is through the "hijacking" of nuclear factors essential for cel-
lular function. Binding of such factors to cisplatin-modified DNA diverts them
from their natural sites, preventing these proteins from performing their critical
tasks. SSRP1 has recently been identified as one component of a heterodimeric
protein complex that facilitates chromatin transcription (FACT), allowing RNA
polymerase II to transcribe through nucleosomes (45). Because SSRP1 binds to
cisplatin-DNA adduets, FACT may recognize the damage through its SSRP1
HMG domain, diverting the protein complex from its normal sites. The result
would be inhibition of transcription elongation by RNA polymerase II. The
repair-shielding and protein-hijacking hypotheses are two possible ways that nu-
clear proteins may trigger cell death following eisplatin damage. In the following
sections we describe recent results that address these hypotheses and introduce
evidence suggesting that other pathways may also contribute to the biological
activity of cisplatin.
II. Kinetic and Thermodynamic Aspects of Protein

Binding to Cisplatin-DNA Adducts
A. Proteins That Bind to Cisplatin-Modified DNA
More than a dozen proteins have been identified that bind to cisplatin-
modified DNA (Table I), recognizing specifically the 1,2-intrastrand crosslink.
SSRP1, the first such protein to be identified, belongs to the HMG family of
DNA-binding proteins. HMG-domain proteins are broadly divided into two
groups, sequence-specific and sequence-neutral (34). Sequence-specific HMG-
domain proteins are usually found only in cells of a particular origin, whereas
sequence-neutral proteins are more widely distributed. The biological func-
tions of HMG-domain proteins are diverse (46) and include chromatin remod-
eling, transcription initiation, and RNA polymerase nucleosome bypass (45).
Essentially all structure-specific HMG-domain proteins bind to cisplatin 1,2-
intrastrand crosslinks. The multifaceted nature of this protein family suggests
that many different critical cellular functions may be interrupted as a result of
HMG protein binding to cisplatin-DNA adducts.
TABLE I
SELECTEDPROTEINSTHAT BIND TO CISPLATIN-MODIFIEDDNA
HMG-
Abbreviation Protein name Function domain
AAG (MPG, ANPG) 3-Methyladenine DNA Repair protein No

glycosylase
H1 Histone linker protein Chromatin structure No
HMG1 High mobility group protein 1 Chromatin structure, remodeling Yes
HMG2 High mobility group protein 2 Chromatin structure, remodeling Yes
Ixrl Intrastrand crosslink Transcription regulation Yes
recognition protein 1
LEF- 1 Lymphoid enhancer-binding Transcription factor Yes
factor 1
RPA (SSB) Replication protein A Nucleotide excision repair No
SRY Sex-determining factor Y Transcription factor Yes
SSRP1 Structure-specific Transcription elongation Yes
recognition protein 1
TBP TATA-binding protein Transcription initiation No
hUBF Upstream binding factor Ribosomal RNA transcription factor Yes
XPA Xeroderma pigmentosum A Nucleotide excision repair No
complementing protein
YB-1 Y-box binding protein 1 Transcription factor No
Reprinted with permissionfrom E. R. Jamiesonand S. J. Lippard, Chem. Rev. 99, 2467-2498 (1999). Copyright©
1999 AmericanChemicalSociety.
In addition to HMG-domain proteins, several other protein classes bind to

cisplatin-damaged DNA. Included are proteins involved in DNA damage recog-
nition, particularly those involved in transcription-coupled NER. An extensive
review of this subject has recently been published (6). Transcription factors,
including YB-1 (47) and the TATA-binding protein (TBP), also bind to cisplatin-
damaged DNA (48, 49). TBP is particularly interesting because the protein is
essential for RNA polymerase II transcription initiation in eukaryotes (50). The
chromatin histone protein H 1, critical for DNA packaging and storage, also binds
to cisplatin lesions (51).
Clearly, many important proteins outside the HMG family also recognize
cisplatin damage, providing even more potential pathways by which the drug
may exert its antitumor effect. Work in our laboratory has focused largely on
the HMG-domain protein family because of the high selectivity for cisplatin
crosslinks and importance in various cellular functions.
B. HMG-Domain Protein Binding to Cisplatin Adducts:

Thermodynamic Considerations
Several studies have been performed to elucidate the strength and speci-
ficity of HMG-domain protein binding to cisplatin-DNA adducts. The majority
of these studies have focused on HMG1, a nuclear protein that contains two
tandem HMG domains designated domain A and domain B (Fig. 3). The func-
tions of HMG1 are not fully understood, but most likely involve activation of
transcription and chromatin remodeling (46).
Electophoretic mobility shift assay (EMSA or bandshift assay) investiga-
tions have revealed many important features of HMG1, HMG1 domain A
(HMGldomA), and HMG1 domain B (HMGldomB) binding to cisplatin
crosslinks. All three proteins bind specifically to 1,2-intrastrand d(ApG) and
d(GpG) adducts, but not to 1,3-intrastrand crosslinks or to interstrand crosslinks.
The affinity of these proteins for cisplatin-DNA adducts is influenced by the se-
quences flanking the crosslink.
In a sequence containing N1G* G'N2, where G'G* represents a 1,2-intra-
strand crosslink, the affinity of HMG-domain protein binding is modulated by
the bases in the N1 and N2 positions (52). The selectivity of binding was ini-
tially examined in 15-bp oligonucleotides, having 9 of the 16 possible flanking
sequences, where N1, N2 = A, C, or T. Synthetic difficulties hampered the prepa-
ration of oligonucleotides containing more than two guanosine nucleosides in a
row, but later efforts provided access to the remaining seven sequences having
either natural guanosine or unnatural 7-deazaguanosine, an isostere that lacks
the reactive N7 nitrogen atom in the purine base, in the flanking positions (53).
This series of experiments reveals several interesting features of HMG-
domain protein binding (Fig. 4). First, the flanking sequence selectivity of
i0 20 30 40 50
HMGIdomA: IMGKGD PKKP~GKMS SYAFFVQTCREEHKKKHPDASVNF S ~F SKKC S ERWKTM
HMGIdomB: 86KKKFKDPNAPKR P___ PSAFFLFC SEYRPKI KGEHP--GLS IGDVAKKLGEMWNNT
LEF-I: 298 H I K K P - - - L N A F M L Y M K E ~ A E C T - -LKESAAINQILGRRWHAL
hSRY: 57QDRVKRP-- -MNAF IVWSRDQRRKMALENP - -RMB_NSEI SKQLGYQWKML
g III
610 710 810
HMGIdomA: SAKEKGK F EDMAKADKARYEREMKTY I PPKGETKKKF
HMGIdomB: A A D D K Q P Y E K K A A K L K E K Y E K D IAAYRAK
LEF-I: SREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKREK NH3 ÷
hSRY: TEAEKWPFFQEAQKLQAMHREKYPNYKYRP C O 2-
1 76 88 164 185 214

box A
I II
I
bo×B -- acidic C-tail
FIG. 3. General structure (lower right) and sequence alignments of several HMG-domain
proteins. The structure is representative of the common a-helical L-shaped motif found in HMG-
domain proteins. Each of the three helices is labeled together with the N and C termini. Sequence
alignments compare four HMG domains: domains A and B of the architectural protein HMG1,
the domain of the transcription factor LEF-1, and the domain of the sex-determining transcription
factor hSRY. A map of the protein HMG1 is shown at the bottom. Adapted with permission from
S. U. Dunham and S. J. Lippard, Biochemistry36, 11428-11436 (1997). Copyright © 1997 American
Chemical Society.
H M G l d o m A is much more p r o n o u n c e d than that of H M G l d o m B . Second,

H M G l d o m A binding is most strongly influenced by the base pair at the N2
position, with A/T base pairs being preferred over C / G base pairs in both the
N1 and N2 positions. Finally, studies using mutant H M G - d o m a i n s show that
base-specific contacts (vide infra) are important for protein affinity, but not se-
quence selectivity. Because H M G - d o m a i n proteins b e n d the helix upon binding
to D N A (54, 55), a preference for A/T over C / G base pairs might be the result of
the increased flexibility of A/T base pairs (56). This conclusion is supported by
thermodynamic studies of AG*G*A and CG*G*C 15-bp oligonucleotides, which
indicate the former duplex to be about 4-fold less destabilized than the latter (57).
C. HMG-Domain Protein Binding to Cisplatin Adducts:

Kinetic Considerations
As indicated previously, H M G - d o m a i n proteins may play an important role
in shielding cisplatin adducts from N E R (43, 44). The ability of H M G 1 to block
0.70
0.60
r [] mmaA|l
0.50
0.40
O
0.30
0.20
0.10
0.0
A,A A,T A,C A,G__T,A T,T T,C T,G C,A C,T C,C C,G G__,AG,T G_,C G,G__
Flanking Bases (N~, N 2)
5'-CCT C T C N I G * G * N 2 T C T TC-3'
3'-GGA G A G N3C C N 4 A G A AG-5'
FIG. 4. Sequence selectivity of HMG-domain proteins for eisplatin adducts determined by
electrophoretic mobility shift assay (EMSA) (52, 53). Normalized quantitation of HMG-domain
binding to cisplatin adducts with different flanking sequences is shown for HMGldomA in open
bars and HMGldomB in filled bars. The sequence of the 15-bp oligonucleotide used for these
studies is shown at the bottom of the figure. G'G* represents a cisplatin 1,2-intrastrand crosslink
and G represents 7-deazaguanosine. (9 represents the ratio of protein-bound DNA to free DNA.
NER requires that it be kinetically and thermodynamicallycompetent to bind to

eisplatin-DNA adduets in eompetition with repair proteins. Bandshift titrations
demonstrate that binding of HMG-domain proteins ean occur with subnanomo-
lar Ka values in vitro, demonstrating high thermodynamic affinity for cisplatin
adduets (52, 58-61). Also, bandshift studies with HMG1 and the repair protein
RPA, both of which independently bind to eisplatin-modified DNA, indicate that
HMG1 bound to eisplatin-modified DNA blocks formation of the RPA complex
(62). One possible explanation for the ability of HMG1 to prevent RPA binding
is that HMG1 more rapidly recognizes and binds to eisplatin-DNA lesions.
Recent stopped-flow experiments (63), using either a fluoreseently labeled
base or an oligonueleotide probe exhibiting fluoreseenee resonance energy trans-
fer (FRET) (64), evaluated the kinetics of HMG-domain binding to cisplatin-
modified DNA. In the latter probe a 20-bp oligonueleotide was labeled with a
fluorescent donor-aeeeptor pair (64). The 5' end of the platinated strand was
109. SETH M. COHEN AND STEPHEN J. LIPPARD
linked to a rhodamine acceptor and a fluorescein donor was installed on the 5'
end of the complementary strand (Fig. 5). These dyes can undergo FRET upon
excitation of the fluorescein at 480 nm, which then undergoes nonradiative en-
ergy transfer to the rhodamine, which in turn can relax by fluorescent emission
(imax = 580 rim). The ratio ofrhodamine to fluorescein emission increases as the
dyes are brought closer together. Because HMG1 binding to cisplatin-modified
DNA increases the bending of the DNA double helix (54), this binding event
brings the dyes closer together. The change in FRET allows for direct monitor-
ing of protein binding, affording both structural and kinetic information. With
the use of a suitable model, changes in the fluorescent lifetime of the fluorescein
donor permit the determination of the helix bend angle.
Coupling of this methodology with stopped-flow techniques provided the
first insight into the kinetics of HMG-domain binding to cisplatin-modified
DNA. The experiments indicate that HMGldomB binds to the cisplatin-
modified FRET probe with a second-order kon value of 1.1 • 0.1 × 109 M-is -1
and a first-order koff value of 30 + 4 s-~. These kinetic data confirm the high
affinity of HMGldomB for cisplatin adducts, the calculated Kd value being
27 ~: 4 nM. More importantly, the results indicate that HMGldomB binds to
cisplatin-modified DNA extremely rapidly, with a rate constant that is near the
diffusion limit. Although the complex is kinetically labile, both kon and koffbeing
[~COOH [~COOH
/ ~ N,,'~,O /'~.N,,~O
H H
Rh= RhodamineAcceptor FI= FluoresceinDonor
5'-Rh-TCT CCT TCT GGT CTC TTC TC 3'

3' - A G A GGA AGA CCA GAG AAG AG-FI-5'
5'-Rh-TCT CCT TCT G*G*T CTC TTC TC - 3'

3' - A G A GGA AGA C C A GAG AAG AG-FI-5'
FIG. 5. Structure of the rhodamine acceptor (top left) and donor fluorescein dyes (top right)
appended to DNA for fluorescence resonance energy transfer (FRET) experiments (64). The se-
quences of the platinated and unplatinated oligonucleotide probes used in the FRET studies are
shown at the bottom of the figure. G* G* represents a cisplatin 1,2-intrastrand crosslink.
rather large, it is clear that HMG-domain proteins can bind rapidly to cisplatin
adducts, as would be required to protect them from repair proteins.
These FRET results were further elaborated by fluorescence studies using a
platinum-modified oligonucleotide probe that contained a nucleoside modified
with a fluorescein tag (63). Seven 16-bp oligonucleotide probes were prepared
containing a single cisplatin 1,2-intrastrand d(GpG) crosslink and a fluorescein-
modified deoxyuridine located at various positions along the platinated strand
(Fig. 6). The largest changes in fluorescence were obtained when the fluorescein-
modified deoxyuridine was placed two bases to the 5' side of the cisplatin adduct.
Larger fluorescence changes occurred with HMGldomA and full-length HMG1
than with HMGldomB.
The experiments with fluorescein-modified deoxyuridine demonstrate that
HMGldomA, HMGldomB, and full-length HMG1 bind to cisplatin-modified
DNA with nearly diffusion-limited kol~values(63). The association rate constant
of HMGldomA is independent of oligonucleotide length, but depends on the
sequences immediately flanking the DNA adduct (Fig. 6). The stopped-flow
Ho.<y o
0 0 ¢,..~-~C00 H
U FL
o o
5 ' - C C T cuFLc TG*G*A C C T TCC-3'

3'-GGA GAG A C C T G G A AGG-5'
5 ' - C C T cuFLc TG*G*T C C T TCC-3'

3 ' - G G A G A G A C C A G G A AGG-5'
5 ' - C C T cuFLc TG*G*C C C T TCC-3'

3 ' - G G A G A G A C C G G G A AGG-5'
FIG. 6. Top: Chemical structure of the fluorescein-modified nucleoside used in fluorescence

stopped-flow kinetics experiments (63). Bottom: Sequence of three of the seven oligonucleotide
probes used to evaluate the kinetics of HMG-domain protein binding to cisplatin-modified DNA.
G* G* represents a cisplatin 1,2-intrastrand crosslink.
104 SETH M. C O H E N AND STEPHEN J. LIPPARD
TABLE II
KINETIC PARAMETERS FOR HMG-DOMAIN PROTEINS BINDING TO CISPLATIN-MODIFIED
PROBES CONTAINING FLUORESCEIN-MODIFIED DEOXYURIDINE (63) (T = 4°C)
Protein Sequence a kon (M-Is -1) koff (s -1) Kd
HMG1 domain A TG*G*A 9.0 4- 2.0 x 107 30 4-11 333 4-134 nM

HMG1 domain A TG*G*T 2.54-1.0 x 107 354-4 1.44-0.6 ttM
HMG1 domain A TG*G*C 4.04-1.0 x 107 264-5 6504-205 nM
HMG1 domain B TG*G*A 1.9 4-1.3 x 107 86 4- 56 453 4- 428 nM
HMG1 domain B TG*G*T 2.4 4-1.5 x 107 73 4- 34 304 4- 237 nM
HMG1 domain B TG*G*C 2.7 4-1.5 x 107 77 4- 50 609 4- 430 nM
%equence shown is only the flanking sequence around the platinum crosslink. The complete sequence
of the 16-bp oligonucleotide probes are given in Fig. 6.
fluorescence studies confirm earlier bandshift results indicating that the base
to the 3' side of the crosslink strongly modulates the affinity of binding for
HMGldomA, and that HMGldomB is substantially less perturbed by the nature
of the flanking sequence (52). In addition, the data suggest that, beyond the bases
directly flanking the lesion, the effect of sequence context is negligible. The
studies reveal that changes in kon, not koff, control the affinity of HMG-domain
proteins for cisplatin-DNA adducts (Table II).
These kinetic studies show that HMG-domain proteins rapidly form
kinetically labile, but thermodynamically stable, complexes with cisplatin-DNA
lesions. The association rate constants are near the diffusion limit, suggesting
that HMG proteins will bind as fast as, or more rapidly than, any other nu-
clear proteins. The results clearly establish that HMG-domain proteins are both
kinetically and thermodynamically competent to shield platinum 1,2-intrastrand
crosslinks from recognition by repair proteins.
III. Structure of an HMG Domain-Cisplatin-DNA

Ternary Complex
A. Cisplatin-DNA Structures
The interpretation of biochemical experiments can be greatly facilitated
by structural information. Once DNA, and more specifically the d(GpG) di-
nucleotide base pair, was established as the primary target for cisplatin, efforts
were made to elucidate both the solution and solid-state structures of cisplatin
1,2-intrastrand crosslinks. The first X-ray structural insights into the nature of
the platinum adduct came with the determination of cisplatin bound to the
dinucleotide d(pGpG) (65). The use of pGpG versus GpG was important be-
cause the former allows a neutral species to form upon platination and facilitates
important hydrogen-bonding interactions that stabilize the complex. The struc-
ture of the dinucleotide adduct confirms that the platinum atom binds to the
N7 nitrogen atoms of the guanine bases (Fig. 7). The bases are oriented in a
head-to-head fashion with both 06 atoms on the same side of the platinum
coordination plane.
Once the core structure of the 1,2-intrastrand adduct was known, further
structural studies focused on longer fragments of DNA. The X-ray structure
of a dodecanucleotide duplex containing a single 1,2-intrastrand erosslink was
solved, revealing how platination affects the global double-stranded DNA struc-
ture (66, 67). This structure displays a large bend in the DNA helix, a shallow
and widened minor groove, and a kink in the DNA at the site of platinum
binding. Surprisingly, the local structure around the platinum atom and the
two bound guanosines is quite different for the dodecanucleotide duplex when
compared with the earlier d(pGpG) structure (vide infra). Several other in-
vestigations have elucidated the solution structure of duplexes modified with
cisplatin, utilizing two-dimensional NMR methods (68) as well as long-range
electron-proton restraints afforded by a nitroxide spin-labeled platinum com-
plex (69). The solution studies confirm the overall features found in the solid
FIG. 7. X-Ray crystal structure of the cisplatin d(pGpG) dinucleotide erosslink (65). Carbon
atoms are shaded in light gray and heteroatoms are shaded in dark gray. The drug is bound to the
dinucleotide by the N7 nitrogen atoms of the guanine bases. The bases are bound in a head-to-
head orientation. A hydrogen bond between a phosphate oxygen and a platinum ammine ligand is
depicted by a dashed line.
FIG. 8. Diagrams of the NMR solution structure (left) and X-ray solid-state structure (right) of
cisplatin-modified DNA duplexes (66-68). The overall structures are similar, demonstrating a bend
in the DNA with a widened, flattened minor groove.
state (Fig. 8), although there are some differences in the specific geomet-
ric parameters of each structure (Table III). Additional studies have afforded
structures of other platinum-modified oligonucleotides including hairpins (70),
1,3-intrastrand crosslinks (71, 72), interstrand crosslinks (73 -75), and most
recently the structure (B. Spingler, D. A. Whittington, and S. J. Lippard,
TABLE III
GEOMETRIC PARAMETERS FROM SEVERAL CISPLATIN-DNA 1,2-INTRASTRAND CROSSLINK
STRUCTURE DETERMINATIONS
Minor groove Dihedral angle a Average helical DNA bend

Structure Method width (A) (deg) twist (deg) (deg) Reference
12-mer duplex NMR 9.4-12.5 47 27 78 68

ll-mer duplex NMR b 9.0-12 58 26 81 69
12-mer duplex X-ray 9.5-11 30 32 39, 55 66, 67
16-mer duplex c X-ray 5.5-12 75 33 61 77
'~Dihedral angle between the cisplatin-modified guanine bases.

blncludes long-range electron-proton distance restraints.
CCisplatin adduct bound by HMGldomA protein.
Reprinted with permission from E. R. Jamieson and S. J. Lippard, Chem. Rev. 99, 2467-2498 (1999). Copyright ©
1999 American Chemical Society.
unpublished data) of a dodecanucleotide modified with the cisplatin analog,

1,2-(R,R)-diaminocyclohexaneplatinum(II) (76).
B. HMGI DomA-Cisplatin-DNA Structure
Although a wealth of structural information is now available for cisplatin-
D N A aclducts, until recently there were no geometric details about their interac-
tions with nuclear proteins. In order to investigate the features dictating H M G -
domain protein recognition of cisplatin-DNA adducts, an X-ray crystal structure
was determined of H M G l d o m A bound to a 16-bp oligonucleotide containing a
single 1,2-intrastrand d(GpG) crosslink (77). The structure was solved by mul-
tiple isomorphous displacement methods using two iodinated derivatives. The
structure confirmed many expected structural traits, but also contained several
surprising features.
The H M G domain binds to the minor groove of the D N A double helix
(Fig. 9) opposite the platinum adduct located in the major groove, an anticipated
finding. The protein is in its normal L-shaped conformation, in good agreement
with an N M R solution structure determination (78). An overlay of the structure
of protein bound to cisplatin-modified D N A (77) and free in solution (78) gives
FIG. 9. X-Ray crystal structure of HMGldomA bound to a cisplatin-modified DNA duplex.

The protein is shown as a light ribbon, the DNA as a darker wire-frame with a ribbon backbone,
and the platinum drug as a light wire-frame rendering. The a helices of HMGldomA are labeled
I, II, and III. The space-filling portion of the figure is the intercalating Phe 37 residue that fills
the hydrophobic notch created between the platinum-modified bases. Note that the protein binds
asymmetrically to the adduct from the minor groove of the DNA duplex. Reprinted with permission
from U.-M. Ohndorf, M. A. Rould, Q. He, C. O. Pabo, and S. J. Lippard, Nature (London) 399,
708-712 (1999).
108 SETH M. COHEN AND STEPHENJ. LIPPARD
an rmsd of 2.1 A, with the largest differences occurring at the C terminus of

helix I, the N terminus of helix II, and the loop between helices I and II (Fig. 9).
The structure of the DNA double helix shows distortions similar to those found in
cisplatin-modified duplexes (66, 68) and to DNA bound by other HMG-domain
proteins (79, 80). Unlike other HMG-domain DNA complexes, however, the
bend in the duplex is not congruent with the angle between helix UII and helix III
of the protein. Instead, H M G l d o m A binds in an asymmetric fashion, biased to-
ward the 3' side of the platinum adduct. The asymmetric positioning of
H M G l d o m A persists in solution, as confirmed in a hydroxyl-radical footprinting
experiment (vide infra). The protein makes several hydrogen-bond, salt-bridge,
and van der Waals contacts with the DNA duplex through helices I and II.
Helix III interacts with the oligonucleotide only through a single water-mediated
contact. A complete contact map for the protein-DNA interactions is presented
in Fig. 10.
The structure of the bound HMG domain reveals an unexpected interca-
lation of the side chain of Phe 37, which is located in the loop region between
51 31
FIG. 10. Contact map showingthe protein-DNA interactions for HMGldomA bound to a
cisplatin-modified16-bpoligonucleotide.Solidlines representhydrogen-bondingcontacts (includ-
ing water-mediated),dashedlines representvan der Waalshydrophobiccontacts,and the triangular
wedge represents intercalationbetweenthe two platinatedbases. Noticethat $41 to the adenosine
3' of the adduct is the onlybase-specificcontact. Reprintedwith permissionfrom U.-M. Ohndorf,
M. A. Rould, Q. He, C. O. Pabo, and S. J. Lippard,Nature (London)399, 708-712 (1999).
helices I and II, into the "hydrophobic notch" in the minor groove formed
between the two cisplatin-bound guanosines. The intercalating residue addition-
ally destacks the adjacent guanines, with a large roll of 61 ° between the bases.
The aromatic phenylalanine side chain forms a n-zr stacking interaction with
the guanine base on the 3' side of the lesion, at a distance of 3.5 ,~, and an edge-
to-face contact with the guanine base on the 5' side of the lesion. This residue is
extremely important for protein binding. Mutation from phenylalanine to trypto-
phan lowers the affinity approximately 5-fold, and replacement by alanine nearly
abrogates protein binding (81).
Another interesting interaction is a base-specific contact between Ser 41
and the N3 nitrogen atom of the adenine base immediately 3' to the platinum
crosslink (Fig. 10). This is the only base-specific contact in the structure, and
mutation of this residue from serine to alanine reduces the affinity of the pro-
tein by 5-fold compared to the wild type protein. The alanine mutant shows
the same flanking sequence selectivity as the wild-type protein (vide supra), in-
dicating that the interaction is important for protein affinity but not specificity
(53). This result is surprising, being the only base-specific interaction and involv-
ing one of the flanking bases, yet the contact does not contribute to sequence
selectivity.
Close inspection of the platinum coordination geometry in the ternary com-
plex reveals the ligand environment to be relaxed relative to that in the structure
in duplex DNA alone (66, 67). Superposition of the platinum center from the
HMG-bound structure and the metal center in the dodecanucleotide duplex
(66, 67) shows striking differences (Fig. 11). Most notably, the dihedral angle
FIG. 11. Superpositionof the platinumcoordinationcenter from the HMGldomA-cisplatin-

DNA ternarycomplexwiththe d(pGpG) structure (right) and the structure of cisplatinbound to a
dodecanucleotide(left).The ternarycomplexis shownin light grayin both overlaydiagrams.The
structures clearlyrevealthat the ternarycomplexis more similarto the d(pGpG)structurethan the
dodecanucleotidestructure. Reprintedwithpermissionfrom U.-M. Ohndorf,M. A. Rould,Q. He,
C. O. Pabo, and S. J. Lippard,Nature (London)399, 708-712 (1999).
between the two bases is "-,30° in the dodecamer structure, but "~75° in the
protein-bound structure. In contrast, comparison of the protein-bound structure
with that of the [Pt(NH3)2{d(pGpG)}] adduct (65) shows a very similar geome-
try, with a dihedral angle in the latter being ~77 °. This analysis suggests that the
platinum center in duplex DNA is in a strained geometry owing to restrictions
imposed by the relatively rigid DNA double helix. After HMG-domain pro-
tein binding, this stress is alleviated, allowing the metal center to take on a more
relaxed geometry with coordination parameters closely resembling those of plat-
inum bound to an unconstrained dinucleotide. The change in geometry lowers
the free energy of complex formation and stabilizes the overall interaction.
C. Mutant HMG-Domain Protein Studies
The detailed molecular structure of the HMGldomA-cisplatin-DNA
ternary complex prompted a series of mutagenesis experiments to elucidate
which interactions modulate protein binding. Some of these mutants, already
mentioned, demonstrate that base-specific contacts are not essential for protein
sequence selectivity and that the intercalating phenylalanine residue is crucial
for high-affinity binding. The primary focus of the additional mutagenesis stud-
ies was to elucidate the significance of the loop region between helices I and II,
and further to explore position 37 occupied by the intercalating phenylalanine
residue. Specifically, the effects on protein affinity and positioning relative to the
TABLE IV
BINDING AFFINITIESAND ORIENTATIONSOF HMG-DOMAIN
PROTEINS WITH CISPLATIN 1,2-INTRASTRANDCROSS-LINKS (81)
Protein Kd (nM) Orientation a
DumA 1.5 -4-0.5 Asymmetric

D u m a AVN 5.0 -4-0.9 n/a
DumA F37W 9.9 + 2.1 n/a
DumA A16F 16.5 + 5.7 Asymmetric
DumA A16F F37A 167 -t- 32 Symmetric
DumA F37A > 1000 rda
DumA $41A 5.8 -4-0.7 rda
DumB 38.7 4- 9.9 Symmetric
DumB iVN 39 4- 2.6 n/a
DumB I37F 19.5 4-13 n/a
DumB F16A 119 -4-39 Asymmetric
DumB 137A 85 4- 17 Symmetric
DumB F16A 137A > 1000 rda
aSymmetric binding displays equal protection, as determined by

hydroxyl-radical footprinting, to bases on both sides of the cisplatin-DNA
1,2-intrastrand crosslink. Asymmetricbinding displays protection to more
of the bases to the 3' side of the cisplatin-DNA 1,2-intrastrand crosslink.
DNA damage were investigated. Six mutants of H M G l d o m A and five mutants

of H M G l d o m B (Table IV) were prepared to address these issues (81).
In the loop region between helices I and II, HMGldomA contains seven
amino acids whereas H M G l d o m B contains only five (see Fig. 3). Because in
H M G l d o m A this loop region contains the critical intercalating Phe 37, it was
speculated that the difference in loop length might explain the higher affin-
ity of H M G l d o m A versus H M G l d o m B for cisplatin-modified DNA (52, 58).
Two mutants were prepared, HMGldomA dxVN and H M G l d o m B iVN, in
which the extra valine and asparagine amino acids were deleted or inserted,
respectively. The affinity of HMGldomA AVN is about 3-fold less than that
of wild-type HMGldomA, and H M G l d o m B iVN demonstrated an affinity for
cisplatin-modified DNA that is essentially unchanged compared to wild-type
H M G l d o m B (Table IV). Bandshift experiments with these mutants indicate
that the length of the loop region between helices I and II is not a critical factor
in determining protein affinity.
Several mutations were made at position 37 in both HMGldomA and
H M G l d o m B to determine the effect on protein affinity. Two position-37 mu-
tants of H M G l d o m A were prepared, H M G l d o m A F37A and HMGldomA
F37W. The tryptophan mutant displays 5-fold lower affinity than the wild-type
protein and the alanine mutant has >1000-fold loss in binding affinity. Two
mutants of H M G l d o m B were also prepared at position 37, H M G l d o m B I37F
and HMGIdomB I37A. The former displays approximately 2-fold higher affinity
than wild-type protein, but H M G l d o m B I37A has about 2-fotd lower affinity
than wild-type HMGldomB. These results suggest that position 37 can also act
as an intercalator for H M G l d o m B and that the native isoleucine residue may
have some intercalative ability. All four of these mutants clearly demonstrate the
importance of intercalation at position 37 in determining protein binding affinity.
Another set of mutant proteins was investigated to evaluate a second possible
intercalating position in helix I at position 16 in domain A. Several sequence-
specific and sequence-neutral HMG-domain proteins utilize intercalators at or
near this site (80). Both single and double mutants were expressed to determine
whether position 16 might be involved in intercalation and how it might affect
protein binding, especially with respect to the influence of the residue at position
37. The mutant H MGldomA A16F has two potential intercalators but decreased
affinity relative to wild-type protein, indicating that in domain A these interca-
lators do not work in a cooperative manner. The double mutant HMGldomA
A16F F37A has lower affinity than HMGldomA, but shows restored binding
relative to H M G l d o m A F37A. This result indicates that, in the absence of an
intercalator at position 37, position 16 can augment binding to cisplatin adducts.
Two mutants o f H M G l d o m B , H M G l d o m B F16A and H M G l d o m B F16A I37A,
show approximately 3-fold and > 1000-fold reduced affinity, demonstrating that
both of these residues contribute to H M G l d o m B binding.
The wild-type proteins and some of the mutants described were also analyzed
by hydroxyl-radical footprinting to determine how the intercalating residues af-
fect the positioning of the protein on the platinated DNA duplex. Two types
of positioning were observed: asymmetric, where the protein preferentially pro-
tects bases to the 3' side of the adduct, and symmetric, where the protein protects
bases equally well on either side of the adduct. In the crystal and in solution,
H M G l d o m A binds in an asymmetric fashion (77). The results of footprinting
studies on the mutant proteins are summarized in Table IV (81). Proteins with an
intercalating residue at position 37 bind asymmetrically, whereas proteins with
an intercalator at position 16 bind in a symmetric fashion. For H M G l d o m A
position 37 is dominant, so that the H M G l d o m A A16F mutant binds in an
asymmetric fashion. These studies demonstrate that intercalating residues in-
fluence both the affinity and orientation of HMG-domain protein binding to
cisplatin-DNA 1,2-intrastrand crosslinks.
IV. Other Consequencesof Cisplatin Treatment of Cells

The role of HMG-domain proteins has been the focus of many investigations
to determine the cytotoxic mechanism of cisplatin (8). Yet, it is quite possible
that other cellular events may contribute to the efficacy of the drug. This sec-
tion describes studies that focus on pathways of cisplatin toxicity not directly
associated with H MG-domain proteins.
A. TelomereShortening
Telomeres are repeating DNA sequences found at the ends of chromosomes
(82). They are responsible for protecting the chromosome from nuclease degra-
dation, end-to-end fusions, and other deleterious events (83-85). Telomeres are
shortened every time cell division occurs, eventually resulting in senescence and
termination of the cell line. In immortalized cells, including cancer cells, telo-
meres do not shorten in a normal fashion following cell division (86). Activation
of the telomerase gene, which encodes for an enzyme involved in maintaining
telomere length, is critical for sustaining cancerous cells (87-90).
The human telomere sequence consists of several thousand nucleotides
of the repeating sequence (TTAGGG)n. This G-rich sequence is potentially
a good target for cisplatin binding. The possibility of preferential platination
of telomere sequences was evaluated by studies of a plasmid comprising ap-
proximately 25% human telomere repeat. These experiments revealed only a
2.6-fold increase in platination of the telomere sequence relative to the re-
mainder of the DNA, a result consistent with a statistical distribution of plat-
inum (91). This finding suggests that if telomeric DNA is selectively targeted
by cisplatin in cells, it probably is not due to the G-rich sequences in telomere
repeats.
H i g h D o s e Cisplatin L o w D o s e Cisplatin
TRF
f 1
AA A A•
3' 3'
w v w I
---- ~ I
R~aI l~aI
Nucleotide Excision | Hint Nucleotide Excision 1 Hinfl
Repair System Repair System
-41.- -4k-
m m
-U-
~ 3' ~ ~ 3 '
Cell cycle arrest Replication and cell division

Replication and cell are completed except telomeres
division are blocked
shortenedTRF
intact
TRF ~ -- ] 3r
-A-- I 1 ~m
~ 3 ' "l" i intactTRF ,
-- Z ~l l 3'
--w--
• cisplatin DNA adduct m ~ sex'and
..~ excised short strand ------ CCC'f~ strand
damaged with cisplafin
FIG. 12. Proposedscheme for dose-dependentcisplatin-mediatedtelomeredamage in HeLa

cell lines. At high doses of cisplatin (left) most cells die before DNA replicationis completeand no
effecton telomerelength (terminalrestrictionfragment,TRF) is observed.At low dosesofcisplatin
(right) cells replicateDNA, but cisplatinadducts cannotbe removedfromtelomeresby nucleotide
excisionrepair (NER). Replicationis blockedbythe DNA adductslocatedat the telomeres,resulting
in incompletereplicationand a shorteningoftelomeres.ReprintedwithpermissionfromT. Ishibashi
and S. J. Lippard,Proc. Natl. Acad. Sci. USA 95, 4219-4233 (1998).
Treatment of HeLa cells with cisplatin results in degradation and shortening

of telomeres in a dose-dependent fashion (92). At high doses, cells die without
exhibiting shortened telomeres. However, at low doses, cells can undergo divi-
sion, with the resulting daughter cells having dramatically shortened telomeres,
which ultimately results in cell death. Cells surviving at least 10 days of exposure
to low levels of cisplatin do not have shortened telomeres. Figure 12 shows a pro-
posed scheme to account for how cisplatin might result in telomere shortening
at the low doses. Although targeting of telomeres by cisplatin is unlikely based
on sequence context alone (vide supra), the possibility remains that telomeres
could be preferentially damaged owing to their unique location and the structure
at the ends of chromosomes (93). Selective damaging of specific regions of the
genome by cisplatin is possible and has been demonstrated at promoters during
activated transcription (94). Because telomeres are not transcribed, they will not
be repaired by the NER system, perhaps making even low levels of telomere
damage sufficient to cause senescence and cell death.
114 SETH M. COHENAND STEPHENJ. LIPPARD
In addition to damaging and shortening the telomeres by directly coordinat-

ing to DNA, several studies indicate that cisplatin can also interfere with telo-
merase activity. Telomerase is a ribonucleoprotein complex that adds "I-TAGGG
repeats to the telomere ends. Although there are non-telomerase-dependent
mechanisms for maintaining telomere length, many cancer cells lines show high
levels of telomerase activity when compared with normal somatic cells, which
display essentially no such activity (86). A study using esophageal cancer cells re-
vealed that cell lines with high levels of telomerase activity were more sensitive
to cisplatin, whereas sensitivity to the anticancer drug 5-fluorouracil showed
no correlation with telomerase activity (95). A study using testicular cancer
cells indicated that cisplatin reduced telomerase activity in a dose-dependent
fashion (96). Other DNA-damaging agents, including doxorubicin, bleomycin,
methotrexate, melphalan, and trans-DDP, had no such effect on telomerase
activity. Evidence for the direct interaction of cisplatin with the telomerase en-
zyme was found in vitro (T. Ishibashi and S. J. Lippard, unpublished data).
Telomerase-active HeLa cell extracts are inactivated by cisplatin in a dose- and
time-dependent manner. In addition, reverse transcriptase polymerase chain
reaction (RT-PCR) shows that cisplatin reacts with the RNA component of the
telomerase enzyme. These studies indicate that cisplatin can affect both telo-
meres and the telomerase enzyme; however, the importance of these interactions
to the overall cytotoxicity of the drug remains to be evaluated.
B. TATA-Binding,H1, and AAG Proteins
Many other important nuclear proteins, which do not belong to the HMG-
domain family, also bind to cisplatin 1,2-intrastrand crosslinks. One example is
the TATA-binding protein (TBP), which is intimately involved in transcription
initiation (50). TBP occurs in eukaryotes that recognize the consensus sequence
T. A. T. a/t. A. a/t (56) located in the promoter region upstream of transcribed
genes. TBP binds to this "TATA box" as a part of a multiprotein complex tran-
scription factor IID (TFIID), thereby initiating a cascade of protein-binding
events leading to transcription by RNA polymerase II and the production of
mRNA. TBP has two domains, a nonconserved N-terminal domain (97) and a
highly conserved C-terminal domain that constitutes the DNA-binding portion.
The conserved C terminus recognizes the TATA box from the minor groove, us-
ing two pairs of intercalating residues to bind and kink the DNA (98, 99). Several
studies show that, both in reconstituted transcription systems and in cells, TBP
can be titrated away from its natural binding site to cisplatin-damaged DNA
(48, 49). Transcription in the reconstituted system can be restored by the ad-
dition of excess TBP. These experiments suggest that platinum-damaged DNA
may divert this critical protein from its natural function.
A recent study focused on the details of the TBP interaction with cisplatin-
modified DNA. The importance of intercalation in the binding of TBP to
platinated D NA was evaluated by investigating a series of oligonucleotide probes

containing a TATAbox flanked by G*G* adducts. The results indicate that, when,
1,2-intrastrand crosslinks were placed at or near the sites of TBP intercalation,
the affinity of the protein for the oligonucleotide increased as much as 175-fold
(100). The binding kinetics demonstrate that the increased affinity is a conse-
quence of a reduction in the dissociation rate constant. These experiments clearly
demonstrate that the intercalating residues in TBP, like those in HMG-domain
proteins, can recognize the hydrophobic notch created by cisplatin adducts,
thereby increasing the stability of the protein-DNA complex. Additional studies
have determined the affinity of TBP for isolated cisplatin adducts, outside the
context of the TATA box (S. M. Cohen and S. J. Lippard, unpublished data).
Preliminary determination of the reverse rate constant by EMSA suggests that
TBP has an affinity for cisplatin adducts comparable to that for the TATA ele-
ment. Most of the approximately 10,000 transcribed genes in mammalian cells
use a TATA box for transcription initiation (50). Tumors from cancer patients
treated with cisplatin contain between 10,000 and 100,000 platinum adducts per
cell (101, 102). Such numbers suggest that TBP could be effectively removed
from its natural binding sites by cisplatin adducts in vivo, resulting in additional
pathways by which the drug might kill cells. Such a pathway would represent a
classic manifestation of the transcription factor hijacking hypothesis.
In addition to transcription factors like TBP, structural proteins in the nu-
cleus have also been shown to bind cisplatin-modified DNA. The histone linker
protein H1 binds to DNA globally platinated with cis-DDP, but not trans-DDP
(51). This protein is an abundant and important nuclear factor that coats DNA-
linking nucleosome core particles. H1 binds to cisplatin-modified DNA even
in the presence of HMG1, suggesting comparable affinities. It is noteworthy
that several chromatin-binding proteins--including H1 which helps determine
structure, SSRP1 which facilitates transcription, and HMG1 which remodels
chromatin--bind specifically to cisplatin-damaged DNA. The involvement of
so many interrelated nuclear factors suggests that the mechanism of cisplatin
genotoxicity could be multifactorial and complex.
Proteins of the NER complex recognize and remove cisplatin adducts from
DNA (6). The 3-methyladenine DNA glycosylase (AAG) family of mammalian
repair proteins also recognizes cisplatin-DNA crosslinks (103). Binding of AAG
may be facilitated by tyrosine intercalation, an interaction the enzyme displays
with other substrates. Although able to bind to cisplatin-DNA adducts, AAG
could not excise the lesions and was incapable of repairing 1, N6-ethenoadenine
(EA) adducts in the presence of cisplatin-damaged DNA. By analogy to tran-
scription factor hijacking, cisplatin may participate in repair factor hijacking, di-
verting AAG from its natural substrates leaving other lesions, such as eA damage,
to mediate cell death. This hypothesis was proposed to explain the synergistic
effects of cisplatin with 1,3-bis-(2-chloroethyl)-l-nitrosourea (BCNU).
116 SETH M. C O H E N AND STEPHEN J. LIPPARD
C. Apoptosisand Ubiquitination
Cisplatin damage to DNA triggers a host of cellular events, ranging from pro-
tein recognition of cisplatin-DNA crosslinks to telomere shortening. A question
remains, however: How do these events contribute to a cascade that leads to cell
death?
Two morphological patterns, necrosis and apoptosis, characterize cell death
(104, 105). Necrosis results when a cell is traumatically damaged, for example, by
puncturing the outer membrane. Apoptosis, coined "programmed cell death"
or "cell suicide," is a controlled pathway that requires new protein synthesis.
Cells exposed to cisplatin exhibit double-stranded DNA cleavage, blebbing of
the cell surface, and cell shrinkage, all of which are consistent with apoptosis
as the means of cell death (106). DNA cleavage produces "-~180-bp fragments,
suggesting internucleosomal scission by an endonuclease. Flow cytometry exper-
iments reveal that cells exposed to cisplatin largely arrest in the second growth
phase (G2) of the cell cycle, indicating that blocked DNA replication, which
occurs in the synthesis phase (S) of the cell cycle prior to G2, is the not the cause
of cell death (107-109). The flow cytometry data are consistent with other ex-
periments demonstrating that cell growth can be inhibited by cisplatin at doses
much lower than those required to inhibit replication (110).
Transcription can also be blocked by cisplatin-DNA adducts. The arresting
of cells in the G2 phase suggests that proteins necessary for mitosis are not
being synthesized, implicating transcription inhibition as the means by which
apoptosis is triggered. Transcription blockage could well be a consequence of
RNA polymerase II stalling at cisplatin-DNA lesions or cisplatin-DNA-protein
ternary complexes. RNA pol II transcription is impeded by UV radiation- or
cisplatin-induced damage leading to phosphorylation on its carboxyl-terminal
domain (111), which results in ubiquitination and degradation in proteasomes
(112). Ubiquitinated proteins are covalently modified by the stepwise activity of
several enzymes (113). This modification signals a targeted response by the cell
and frequently results in degradation of the modified protein inside proteasomes.
Other forms of DNA damage do not elicit this cascade. UV radiation-induced
reduction in RNA pol II activity is alleviated in repair-competent cells, but not
in repair-deficient xeroderma pigmentosum cell lines (114).
These observations suggest one possible mechanism for cytotoxicitywhereby
cisplatin treatment results in transcription inhibition and degradation of RNA
pol II owing to ubiquitination. RNA pol II levels cannot be restored because
shielding by HMG-domain proteins, or other proteins that have a high affin-
ity for cisplatin-DNA adducts, protects cisplatin lesions from nucleotide exci-
sion repair. As a consequence, there is a continual decline in RNA pol II and
transcriptional activity, which ultimately may lead to apoptosis. Although more
experiments are required to explore this hypothesis, it is an intriguing scenario
that incorporates both the phenomena of transcription inhibition by and repair

shielding of cisplatin-DNA cross-links.
Other possible pathways need to be explored to determine how cisplatin
leads to apoptosis. In addition to ubiquitination and shortening of telomeres,
the relationship between cisplatin and p53 has not been resolved. The latter
subject has been thoroughly discussed in a recent review (6).
V. Developmentof New Platinum Compounds

Although cisplatin and carboplatin (vide infra) have been very successful for
the treatment of cancer, the compounds are not ideal drugs. Neither is orally
active and both must be administered intravenously. Cisplatin has several detri-
mental side effects, including nephrotoxicity, neurotoxicity, and severe nausea
(3). Patients also experience both inherent and acquired resistance to the drug
(115, 116). As a consequence of these shortcomings, several research efforts have
focused on producing compounds with equal or greater potency, lower toxicity,
efficacy against cisplatin-resistant tumors, and oral availability (3). In the fol-
lowing section we discuss novel discovery methods and a better understanding
of cisplatin activity which are leading the way toward developing new platinum
drugs and chemotherapeutic regimens.
A. Mononuclear PlatinumComplexes
To date, the FDA has approved only one cisplatin analog. Carboplatin (Figs. 1
and 13) has a 1,1-dicarboxylatocyclobutane unit as the labile ligand replacing the
two chloride ions in cisplatin. This chelating dicarboxylate dianion dissociates
more slowly than the chloride ligands, resulting in a less reactive compound
with substantially diminished side effects. Because of the reduced toxicity, car-
boplatin has been used very successfully and is gradually replacing cisplatin in the
clinic. Although not approved in the United States, oxaliplatin is used in parts of
South America, Asia, and Europe (117). In this compound, the ammine ligands
of cisplatin are replaced by cis-l,2-(R,R)-diaminocyclohexane and the chloride
groups by a chelating oxalate leaving group. The compound has been pursued
owing to its efficacy against colorectal cancer and some cisplatin-resistant tumors
(118-130).
Some Pt(IV) compounds are being investigated because of their oral activity.
These compounds, designated JM216 and JM221, are octahedral platinum(IV)
complexes that are reduced in vivo to square planar platinum(II) compounds
with kinetically inert ammine and cyclohexylamine ligands and a pair of labile
chloride leaving groups (3). The resulting platinum(II) metabolites then react
in a fashion analogous to cisplatin. These compounds are currently undergoing
H2
N~ /sO 0
\/ , ~
N2
CarboplaUn Oxallplatln
o o
.,N\I/°. .3N\I/°,
/N -
o 0
JM216 JM221
FIG. 13. Chemical structures of several mononuclear platinum compounds that have been
investigated as anticancer drugs. The octahedral compounds (bottom) are Pt(IV) and are intended
as orally active alternatives to cisplatin. Of the compounds shown, only carboplatin (upper left) is
approved for use in the United States.
clinical trials and may alleviate the need for intravenous administration of plat-
inum drugs. The effect of the cyclohexylamine on the efficacy has not been fully
resolved, however.
Because JM216, JM221, and oxaliplatin have demonstrated some promise
against cisplatin-resistant cell lines, the biological effects of different amine
ligands have been investigated. Several studies have demonstrated that such
spectator or carrier ligands can affect the ability of a platinum adduct to block
replication by various DNA polymerases (121-125). Apparently, HMG-domain
proteins, including HMG1 and human upstream binding factor (hUBF), bind
to oxaliplatin and JM216/221 lesions with lower affinity than to cisplatin-DNA
adducts (123, 126). This difference suggests that HMG-domain proteins may
not play a critical role in the genotoxicity of oxaliplatin and JM216/221, thereby
explaining the lack of cross-resistance with cisplatin-resistant cell lines. More
studies are required to elucidate the effects of various spectator ligands and the
pathways that might mediate the toxicity of these complexes.
Apart from the compounds described above, few cisplatin analogs have pro-
gressed through stage-three clinical trials (3). As an alternative to the conven-
tional design of platinum drugs, which contain a pair ofcis leaving groups, several
mononuclear compounds with trans positioning of the ligands have been pursued
as possible drug candidates. Many such compounds use aromatic amines as the
inert spectator ligands (3). These compounds have met with mixed success,
showing some utility against cisplatin-resistant cell lines. To date, no mononu-
clear trans platinum compound has made significant progress in the clinic.
B. Polynuclear Platinum Complexes

As an alternative paradigm to mononuclear platinum compounds, several
polynuclear platinum complexes have been prepared for use as antitumor drugs
(Fig. 14). Compounds containing two, three, and four platinum centers (•27)
in both trans and cis configurations have been investigated (128-131). Of these
compounds, monofunetional complexes in which the leaving groups are trans
to the linking ligand have received the greatest attention. Recently, one in par-
ticular, BBR3464, a trinuclear bifunetional platinum compound (Fig. 14), has
entered phase I clinical trials (132). These compounds form a variety of DNA
adduets, with 1,3- and longer-range interstrand erosslinks being the major ones
(128). Not surprisingly, these lesions are not tightly bound by HMG-domain
proteins (vide supra), indicating that their toxicity, like that of oxaliplatin and
-•2+ ~ 2+
H3N~ /Cl Cl~ /NH3 Cl~ /NH3 H3N"\ //el
/Pt Pt /Pt Pt
N /N
H3N NH2(CH2)6H2N NH3 H3N ~NH2(CH2)6H2
N/ ~NH3
1,1/C,C 1,1/t,t
CI,~ /NH3 H3NN /CI Cl~ ,/NH3 H3N~ ,/C----II1+

.Pt Pt /Pt Pt
CI / \NH2(CH2)sH2
N/ ~CI H3N ~NH2(CH2)6H2
N/ ~Ct
2,2/c,e 1,2/t,c
Cl~ /NH3 H3NN /NH2(CH2)6H2N~/NH3--] 4+

/et et /Pt
H3N ~NH2(CH2)6H2
N/ ~NH3 H3N ~CI
1,0,1/t,t,t
FIG. 14. Chemical structures of several multinuelear platinum compounds that have been
investigated as anticancer drugs. The naming scheme indicates the number of leaving groups on
each metal center ( I, 2, etc.) followed by the stereochemistry of each metal center (eis = e, trans = t).
The trinuclear compound (bottom) is BBR3464, which is in phase I clinical trials.
JM216/221, is most likely mediated by a different pathway from that of cisplatin

(133). The potential utility of these compounds in practical medicine is currently
uncertain.
C. Combinatorial/Parallel SynthesisApproaches
Combinatorial and parallel synthetic approaches have assumed a promi-
nent position in drug discovery. Such efforts initially focused on peptide-based
drugs, and subsequently combinatorial synthesis was adapted for organic small
molecule discovery (134, 135). For inorganic synthesis, combinatorial methods
have been applied to the discovery of solid-state materials (136, 137) and catalyst
design (138).
Recently, we have applied parallel synthesis to platinum drug discovery (139).
Conceptually, this process should also be useful for the discovery of inorganic
drugs (140) of various types, such as imaging agents (141) and antimalarials (142),
although the focus of our work was on platinum anticancer compounds. Our gen-
eral synthetic scheme and robotic apparatus for parallel synthesis of cisplatin
analogs are depicted in Fig. 15. The synthesis allows for the variation of both the
spectator and leaving group ligands. In a 96-well reaction block, either K2PtC14
or K[Pt(NH3)C13] is activated with KI, forming [PtI4]2- or [Pt(NH3)I3]-, re-
spectively. Subsequently, the spectator ligand(s) are added followed by a slight
excess of AgNO3, which precipitates AgC1 and AgI. The soluble products are
then filtered into vials, located below the reaction block, containing various
leaving-group ligands. The samples are lyophilized and analyzed by atomic ab-
sorption (AA) spectroscopy for platinum content. In several months, roughly
3600 reactions were run and the products screened for their ability to inhibit
transcription (vide infra). To put this value in perspective, approximately 3000
platinum compounds had been synthesized and evaluated for activity in the en-
tire chemical literature prior to this study (3). Various spectator ligands were
examined, including aliphatic amines, aromatic amines, phosphines, and sulfur-
containing compounds. Also, several types of leaving groups were screened,
including chlorides, monodentate acids, and bidentate acids.
Of the 3600 reaction mixtures, 14 hits were obtained and further evalu-
ated in a concentration-dependent manner. Of these 14, four displayed good
activity in the concentration-dependent study. Of the four compounds,
cis-[ (isopropylamine)2PtC12], cis-[(cyclobutylamine)2PtC12], and cis-[ammine
(cyclobutylamine)PtC12] had been previously identified as potential drug candi-
dates (143-145). The fourth compound, cis-[ammine(2-amino-3-picoline)PtC12],
represents a novel lead in platinum drug discovery, bearing some resemblance
to recently investigated platinum compounds (3, 146, 147).
The parallel synthesis of platinum compounds is an alternative approach to
the discovery of new anticancer drugs. The methodology can be easily adapted
to produce other kinds of compounds, and some having trans geometries have
cl\ /cl 4KI I\ /~ 2L L\ /I
Pt • • Pt
CI/ XCI I/%1 L/ \ l
L~ ~1 8AgNO3 L~ ~ OH2 xsZ L~ /Z

Pt Pt Pt
L/ \, L/ \Z
ReactionBlock
Co~l~~tionVial
FIG. 15. Synthetic scheme (top) and apparatus (bottom) for parallel synthesis of platinum
drug leads. Tetrachloroplatinate(II) is activated with KI, followed by addition of 2 equivalents of
spectator ligand (L). Silver chloride is then added, precipitating AgCI and AgI and producing the
aquated platinum species. The soluble aqua complex is t~ltered from the reaction block, through a
Teflon flit, and into a collection vial containing leaving-group ligands (Z). The samples are lyophilized
and analyzed for platinum content by atomic absorption (AA) spectroscopy. In approximately one
year, 3600 reactions were performed by using this methodology. The compounds were tested against
HeLa cell lines using a fluorescent dye reporter assay.
already been prepared as a proof of concept (139). A parallel synthesis effort

requires an accurate and rapid screening process in order to be successful. The
next section discusses the high-throughput screens used to evaluate these new
platinum drug candidates.
D. Screenin9 Methods
To evaluate the large number of compounds produced by parallel synthesis,
a rapid, mechanism-based assay was devised. Cisplatin, unlike many other DNA-
damaging agents, causes a dose-dependent diminution in transcription activity
(148). To identify new drug candidates that kill cells by a related mechanism, an
assay was developed to screen for inhibition of transcription in Jurkat and HeLa
cell lines. The assay is based on CCF2/AM molecules containing fluorescein,
coumarin, and/~-lactam components (Fig. 16) (149). The CCF2/AM dye dif-
fuses passively through cellular membranes, whereupon intracellular esterases
remove several protecting groups, resulting in a charged species that is trapped
inside the cell. Excitation of the coumarin moiety at 409 nm results in FRET to
the fluorescein, which emits light at 520 nm and gives the cells a green color. If
the cells are expressing a fl-lactamase, the linker is cleaved, separating the two
dyes and preventing FRET. With the molecule cleaved, the coumarin dye emits
at 447 nm, giving the cells a blue color. By monitoring the ratio of green and
blue cells, a readout for measuring the inhibition of gene expression is obtained.
Screening for transcription inhibition in this manner requires approximately
28 h and allows for the analysis of 72-96 compounds per day without robotics.
BlaM HeLa cells, stably transfected with a vector encoding for TEM-1
fl-lactamase from E. coli, were placed in 96-well plates. The cells were treated
with different platinum compounds and then exposed to the CCF2/AM dye.
A ratiometric response was obtained by using a fluorescent plate reader mon-
itoring at 530 nm and 460 nm. Compounds that demonstrated a 530:460 ratio
greater than that of cisplatin controls were further evaluated in a concentration-
and time-dependent fashion (139).
E. Steroid Hormones, Cisplatin, and Cancer

In addition to discovering new platinum compounds with genotoxic activ-
ity, the knowledge gained about the mechanism of action of cisplatin has also
been used to develop new combination therapies for increasing its efficacy.
A recent report demonstrated that HMG1 mRNA levels were upregulated by
estrogen treatment of MCF-7 breast cancer cells (150). This finding allowed us to
examine directly the postulated role of HMG1 in mediating cisplatin cytotoxicity
(151).
Treatment of MCF-7 breast cancer cells with estrogen or progesterone up-
regulates HMG1 levels, as demonstrated by immunofluorescence. Treatment
of MCF-7 cells with either estrogen or progesterone followed by exposure to
BIOCHEMISTRY OF CISPLATIN 19,3
B,oy.~o.~o, ,oo.~y.O.~y.oAo
c,~.~.~ ~
O T v CCF2/AM
CO2AM
/ / a
"O.,.f-~,/O...~O ,~V.y.~.~,., 7°°'_\
GreenLight \
/
/ ° °~"NH s. ilLA
409 nm ~-I.a~amase
\ o,~% + ~-#. /
\ o o.-~.. ~.~.co2
\ .o.c.~S~ cd /
°02-
FIG. 16. Scheme of the fluorescent dye system used to screen fnr new platinum anticancer
drug candidates. Ceils expressing l%lactamases cleave CCF2/AM and the cells appear blue from
the coumarin emission at 447 nm. The fluorescence of the cleaved fluorescein unit is quenched due
to the free thiol group. If genotoxic agents are blocking transcription of ~-lactamases, however, the
dye cannot be cleaved and undergoes FRET with an emission from the fluorescein dye at 5"20nm,
giving the cells a green color. Transcription'a] activity can be calculated ratiometrically from the green
versus blue emission. Reprinted with permission from E. R. Jamieson and S. J. Lippard, Chem. Rev.
99, 2467-2498 (1999). Copyright © 1999 American Chemical Soeie~.
cisplatin causes a 2-fold i n c r e a s e in sensitivity to the d r u g (Fig. 17). C o t r e a t -

m e n t with half an e q u i v a l e n t each o f e s t r o g e n a n d p r o g e s t e r o n e gives a
synergistic effect, r e s u l t i n g in a 4-fold sensitization to cisplatin. A 2-fold sen-
sitization t o w a r d c a r b o p l a t i n also can b e achieved, b u t r e q u i r e s that the cells b e
p r e t r e a t e d the p l a t i n u m c o m p o u n d owing to the slower kinetics o f D N A binding.
100
"~ 10-.
--- none
A progesterone ]J "1~
o both ± 1
1
[cisplatin] (laM)
100
10-.
none
1 ~ ~ 1~ 116 210
[carboplatin] (pM)
FIG. 17. Cytotoxieity assay plots demonstrating sensitization of MCF-7 breast cancer cells to
cisplatin resulting from steroid hormone treatment (151). The top plot shows that the cells were
~2-fold more sensitive to cisplatin when exposed to estrogen or progesterone and ~4-fold more
sensitive when subjected to both steroid hormones. The bottom plot shows that a similar sensitization
was also obtained with carboplatin, although a different regimen was required because of its slower
reaction kinetics. No sensitization was seen for trans-DDP or with steroid hormone receptor-
negative HeLa cells, consistent with HMG1 upregulation being the source of sensitization (data not
shown). Reprinted with permission from Q. He, C. H. Liang, and S. J. Lippard, Proc. Natl. Acad.
Sci. USA. 97, 5768-5772 (2000).
No sensitization toward trans-DDP or calieheamicin, another cytotoxic agent,

is obtained upon steroid hormone treatment. This work illustrates how a bet-
ter understanding of the mechanism of cisplatin can lead to potentiation of its
activity. By knowing specific biochemical interactions at the molecular and cel-
lular levels, a strategy was devised that could predictably enhance the activity
of the drug. Cisplatin, estrogen, and progesterone are all FDA-approved drugs.
A phase I clinical trial combining progesterone-carboplatin chemotherapy for
women suffering from ovarian cancer has recently commenced (unpublished).
VI. Concluding Remarks

The success of cisplatin for treating cancer is unrivaled in the history of
inorganic medicinal chemistry. After a serendipitous discovery, years of chemical
research have begun to reveal how the drug elicits its genotoxic and cytotoxic
effects. The information gathered about the mechanism of action is now being
used to develop new treatment strategies and discover new platinum drugs.
Despite great strides in this field, many questions remain. Although a strong
body of evidence supports the involvement of HMG-domain proteins in mediat-
ing cisplatin efficacy, exactly how these proteins trigger cytoxicity remains to be
determined conclusively. HMG-domain sensitization may involve both repair-
shielding and protein-hijacking mechanisms. The roles of other nuclear proteins,
such as TBP and AAG, and the precise nature of the interaction of these proteins
with cisplatin-damaged DNA have yet to be elucidated. The effects of telomere
damage and the importance oftelomere maintenance represent another intrigu-
ing route by which cisplatin may ultimately cause cell death. Tile question of why
cisplatin is particularly effective against testicular cancer remains unanswered.
The pursuit of these remaining questions is critical to understanding one
of the most successful anticancer drugs to date. More detailed insights into the
mechanism of cisplatin will continue to provide new clues for fighting cancer
and perhaps to fulfill the promise of developing other inorganic drugs that rival
cisplatin efficacy with reduced side effects. The story of this fascinating drug
clearly has many more interesting lessons to teach to bioinorganic and medicinal
chemistry communities.
ACKNOWLEDGMENTS
The authors thank Q. He, Dr. T. Ishibashi,Dr. E. R. Jamieson,Dr. U.-M.Ohdorf,A.E Silverman,

Dr. B. C. Spingler, and Dr. C. J. Ziegler for permission to cite unpublished data. The research
undertaken in our laboratoryand describedhere was supported by grants from the National Cancer
Institute (grant CA 3499"2).S. M. C. is the recipient of a National Institutes of Health postdoctoral
fellowship.
126 SETH M. COHEN AND STEPHEN j. LIPPARD
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Cisplatin: From DNA Damage

Cisplatin [c/s-DDP, cis-diamminedichloroplatinum(II)] is a potent antieancer

1Author to w h o m correspondence should be addressed. Telephone: (617) 253-1892. Fax: (617)

Progressin NucleicAcid Research Copyright© 2001 by AcademicPress.

efforts to understand the mechanism of genotoxicity of cisplatin are expected

Cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] is a relatively simple

H3N CI H3N 0% "'/ CI/ ~NH3

cisplatin carboplatin trans-DDP

[Cl']intracellular = -2-30 mM Aquation

DNA H3N~ /OH2~ 2+ RNA

II. Kinetic and Thermodynamic Aspects of Protein

AAG (MPG, ANPG) 3-Methyladenine DNA Repair protein No

In addition to HMG-domain proteins, several other protein classes bind to

B. HMG-Domain Protein Binding to Cisplatin Adducts:

1 76 88 164 185 214

H M G l d o m A is much more p r o n o u n c e d than that of H M G l d o m B . Second,

C. HMG-Domain Protein Binding to Cisplatin Adducts:

NER requires that it be kinetically and thermodynamicallycompetent to bind to

Rh= RhodamineAcceptor FI= FluoresceinDonor

5'-Rh-TCT CCT TCT GGT CTC TTC TC 3'

5'-Rh-TCT CCT TCT G*G*T CTC TTC TC - 3'

5 ' - C C T cuFLc TG*G*A C C T TCC-3'

5 ' - C C T cuFLc TG*G*T C C T TCC-3'

5 ' - C C T cuFLc TG*G*C C C T TCC-3'

FIG. 6. Top: Chemical structure of the fluorescein-modified nucleoside used in fluorescence

Protein Sequence a kon (M-Is -1) koff (s -1) Kd

HMG1 domain A TG*G*A 9.0 4- 2.0 x 107 30 4-11 333 4-134 nM

III. Structure of an HMG Domain-Cisplatin-DNA

Minor groove Dihedral angle a Average helical DNA bend

12-mer duplex NMR 9.4-12.5 47 27 78 68

'~Dihedral angle between the cisplatin-modified guanine bases.

unpublished data) of a dodecanucleotide modified with the cisplatin analog,

FIG. 9. X-Ray crystal structure of HMGldomA bound to a cisplatin-modified DNA duplex.

an rmsd of 2.1 A, with the largest differences occurring at the C terminus of

FIG. 11. Superpositionof the platinumcoordinationcenter from the HMGldomA-cisplatin-

Protein Kd (nM) Orientation a

DumA 1.5 -4-0.5 Asymmetric

aSymmetric binding displays equal protection, as determined by

DNA damage were investigated. Six mutants of H M G l d o m A and five mutants

IV. Other Consequencesof Cisplatin Treatment of Cells

Cell cycle arrest Replication and cell division

FIG. 12. Proposedscheme for dose-dependentcisplatin-mediatedtelomeredamage in HeLa

Treatment of HeLa cells with cisplatin results in degradation and shortening

In addition to damaging and shortening the telomeres by directly coordinat-

platinated D NA was evaluated by investigating a series of oligonucleotide probes

that incorporates both the phenomena of transcription inhibition by and repair

V. Developmentof New Platinum Compounds

B. Polynuclear Platinum Complexes

CI,~ /NH3 H3NN /CI Cl~ ,/NH3 H3N~ ,/C----II1+

Cl~ /NH3 H3NN /NH2(CH2)6H2N~/NH3--] 4+

JM216/221, is most likely mediated by a different pathway from that of cisplatin

L~ ~1 8AgNO3 L~ ~ OH2 xsZ L~ /Z

already been prepared as a proof of concept (139). A parallel synthesis effort

E. Steroid Hormones, Cisplatin, and Cancer

cisplatin causes a 2-fold i n c r e a s e in sensitivity to the d r u g (Fig. 17). C o t r e a t -

No sensitization toward trans-DDP or calieheamicin, another cytotoxic agent,

VI. Concluding Remarks

The authors thank Q. He, Dr. T. Ishibashi,Dr. E. R. Jamieson,Dr. U.-M.Ohdorf,A.E Silverman,

1. S. J. Lippard, Science 218, 1075-1082 (1982).

68. A. Gelasco and, S. J. Lippard, Biochemistry 37, 9230-9239 (1998).

136. E. Danielson, J. H. Golden, E. W MeFarland, C. M. Reaves, W. H. Weinberg, and X. D. Wu,

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5'-Rh-TCT CCT TCT GGT CTC TTC TC - 3'

5 ' - C C T cuFLc TGGA C C T TCC-3'

5 ' - C C T cuFLc TGGT C C T TCC-3'

5 ' - C C T cuFLc TGGC C C T TCC-3'

HMG1 domain A TGGA 9.0 4- 2.0 x 107 30 4-11 333 4-134 nM