[CANCER RESEARCH 63, 100 –106, January 1, 2003]
A Nuclear Protein Complex Containing High Mobility Group Proteins B1 and B2,
Heat Shock Cognate Protein 70, ERp60, and Glyceraldehyde-3-Phosphate
Dehydrogenase Is Involved in the Cytotoxic Response to DNA Modified
by Incorporation of Anticancer Nucleoside Analogues1
Eugene Y. Krynetski, Natalia F. Krynetskaia, Marco E. Bianchi, and William E. Evans2
Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 [E. Y. K., N. F. K., W. E. E.]; University of Tennessee, Memphis,
Tennessee 38163 [E. Y. K., N. F. K., W. E. E.]; and San Raffaele Scientific Insitute, via Olgettina 58, 20132 Milano, Italy [M. E. B.]
ABSTRACT
Thiopurine treatment of human leukemia cells deficient in components
of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate
the cellular sensor(s) responsible for recognition of DNA damage in cells
with an inactive mismatch repair system, we isolated a multiprotein
nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins
1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide
isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The
same complex was also shown to bind synthetic oligodeoxyribonucleotide
duplexes containing the nonnatural nucleosides 1-␤-D-arabinofuranosylcytosine or 5-fluoro-2ⴕ-deoxyuridine. Fibroblast cell line derived from
Hmgb1ⴚ/ⴚ murine embryos had decreased sensitivity to thiopurines, with
an IC50 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and
exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not
incorporated into DNA. These findings indicate that the HMGB1HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA
modifying chemotherapy.
INTRODUCTION
Induction of apoptosis in malignant cells by damaging DNA is a
common mechanism of cancer chemotherapy (1). DNA lesions caused
by irradiation or chemical modification of DNA, trigger the apoptotic
machinery and thereby eliminate cells with competent mechanisms
for recognizing DNA damage and carrying out apoptosis.
Treatment of cells with DNA-reactive compounds (e.g., cisplatin)
or structural analogs of DNA precursors (e.g., cytarabine, fludarabine,
and thiopurines) results in growth arrest and apoptosis (2– 4). Although major components of the apoptotic machinery have been
elucidated, the molecular interaction between damaged DNA and
cellular processes that initiate apoptosis remain obscure. Recent studies demonstrated that distinct structural elements of damaged DNA
provide the sites for binding of DNA damage-recognizing proteins
and subsequent assembly of multiprotein complexes, which normally
maintain the integrity and fidelity of genetic information (4 – 6). The
postreplicative MMR3 system is another example of protein comReceived 2/12/02; accepted 10/30/02.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported, in part, by Grant R37 CA36401 from the National
Institutes of Health, Cancer Center Support Grant CA 21765, and by the American
Lebanese Syrian Associated Charities.
2
To whom requests for reprints should be addressed, at St. Jude Children’s Research
Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794. Phone: (901) 4953663; Fax: (901) 525-6869; E-mail: [email protected].
3
The abbreviations used are as follows: MMR, mismatch repair; MP, mercaptopurine;
dGS, 2⬘-deoxy-6-thioguanosine; PI, propidium iodide; TdT, terminal deoxynucleotidyl
plexes involved in recognition of DNA damage after anticancer therapy, and the binding of MMR proteins to modified DNA has been
shown in in vitro experiments (7, 8).
Cytotoxic effect of thiopurines, a class of widely used antileukemic
agents (9), is achieved through a common mechanism that exploits the
conversion of these drugs into deoxythioguanosine triphosphate, with
subsequent incorporation into DNA (8, 10, 11). In contrast to ␥-irradiation, such incorporation induces local structural changes in DNA
(12) rather than destroy its integrity and affects DNA-protein interactions, including topoisomerase II and RNase H (13, 14). This
mechanism may also contribute to the association of thiopurine treatment with an increased risk of therapy-related leukemia or brain
tumors, when thiopurines are used concurrently with topoisomerase II
inhibitors or irradiation (15, 16).
MMR proteins have been demonstrated to modulate thiopurine
cytotoxicity, and the loss of MMR activity can confer a resistance
phenotype to thiopurine treatment (17). However, several human and
murine cells deficient in MSH2, a key component of MMR, demonstrated high sensitivity to anticancer drugs, including thiopurines (13,
18). These paradoxical observations indicate the existence of complementary biochemical pathways that activate cell death in response to
DNA damage in MMR-deficient cells.
To further understand the mechanisms by which thiopurines exert
their cytotoxic effects, we investigated how incorporation of thiopurines into DNA is recognized by cellular machinery in human MSH2deficient acute lymphoblast leukemia cells. Characterization of this
process and identification of sensor proteins in these cells will provide
new insights into the determinants of drug sensitivity in cancer cells
and may facilitate the development of more effective and less toxic
anticancer regimens. Here we present new findings indicating that
DNA surveillance mechanisms identify chemical modification of DNA
by detecting nonnatural nucleosides with an HMGB1-containing multiprotein complex. Furthermore, we report the isolation and identification
of components of a nuclear multiprotein complex that has high affinity to
duplex DNA modified by incorporation of dGS, ara-C, or 5FdU and show
that murine fibroblasts deficient in one component of the complex,
Hmgb1, are 10-fold more resistant to thiopurine treatment than their
wild-type counterparts.
MATERIALS AND METHODS
Cell Culture and Preparation of Nuclear Extracts. The human B-lineage
leukemia cell line Nalm6 was obtained from the DSMZ-German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were
grown in complete RPMI 1640 (BioWhittaker, Walkersville, MD), which
transferase; TUNEL, Tdt-mediated nick end labeling; BrdUrd, bromodeoxyuridine; ara-C,
1-␤-D-arabinofuranosylcytosine; 5FdU, 5-fluoro-2⬘-deoxyuridine; EMSA, electrophoretic
mobility shift assay; HSC70, heat shock cognate protein 70; ERp60, protein disulfide
isomerase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PVDF,
polyvinylidene difluoride; HMGB1, high mobility group protein B1; HMGB2, high
mobility group protein B2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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contained 10% fetal bovine serum and 2 mM L-glutamine, at 37°C in an
atmosphere of 5% CO2. The doubling time of Nalm6 cells under these
conditions was ⬃19 h. The number and viability of the cells were determined
in duplicate by trypan-blue exclusion, using a Burker-Turk chamber. In all
experiments, the initial concentration of cells was 0.25 ⫻ 106/ml. A single dose
of 6-MP, which was dissolved in medium, was added to the culture medium so
that the final concentration was 10 ␮M (determined by measurement of the
absorbance of the medium; wavelength, 320 nm; extinction coefficient,
19,600). Cytotoxicity was expressed as the ratio of the number of viable cells
incubated with MP (i.e., cell count ⫻ percentage of viable cells) to the number
of viable cells in the control medium, which lacked MP (i.e., cell count ⫻ percentage of viable cells). Nuclear extracts were prepared according to the
method of Dignam et al. (19) Protein concentration was determined by
Bradford dye-binding procedure using Bio-Rad Protein assay (Bio-Rad, Hercules, CA).
Cell Cycle Synchronization. Nalm6 cells were seeded at a concentration
of 1 ⫻ 106/ml in complete RPMI 1640 that contained 2 mM thymidine (Sigma,
St. Louis, MO). The culture was then incubated at 37°C for 16 h. Cells were
washed twice with PBS and centrifuged at 500 ⫻ g for 10 min at room
temperature. Cells were resuspended in 200 ml of RPMI 1640, and 2 h after the
removal of thymidine, MP was added to 20 ml of the cell suspension so that
the final concentration of the drug was 10 ␮M.
DNA Modification. The level of dGS incorporation into DNA after MP
treatment was determined by high-performance liquid chromatography analysis by methods we have previously described in detail (13).
Cell Cycle Analysis, TUNEL Assay, and Caspase Activation Assay. For
cell cycle analysis, cells were centrifuged and resuspended at a concentration
of 1 ⫻ 106/ml in a PI staining solution (0.05 mg/ml PI, 0.1% sodium citrate,
0.1% Triton X-100). Each sample was treated with DNase-free RNase (5
ng/ml; Calbiochem, San Diego, CA) at room temperature for 30 min, filtered
through 40-␮m mesh, and analyzed by a FACScan flow cytometer (Becton
Dickinson, San Jose, CA) collecting the fluorescence (wavelength range,
563– 607 nm) from PI-bound DNA (⬃15,000 cells). The percentages of cells
within each phase of the cell cycle were computed by using ModFit software
(Verity Software House, Topsham, ME).
For TUNEL analysis of apoptosis, 1 ⫻ 106-3 ⫻ 106 fixed cells were end
labeled with brominated dUTP (BD Biosciences, San Jose, CA) by using TdT
(Roche Diagnostics, Indianapolis, IN). The samples were then incubated with
FITC-conjugated anti-BrdUrd monoclonal antibody (BD Biosciences) for 1 h
at room temperature. After cellular DNA was counterstained with PI, the cells
were treated with RNase and analyzed by a FACS Calibur flow cytometer
(Becton Dickinson). Matched aliquots of each sample were treated identically,
except one aliquot of each pair was not incubated with TdT during the
end-labeling procedure. Thus, aliquots that lacked TdT served as an internal
negative control for FITC fluorescence of each sample. Cells exhibiting FITC
fluorescence greater than background levels were classified as apoptotic.
For assays of caspase activation, ⬃3 ⫻ 105 cells were incubated with the
caspase inhibitor FAM-VAD-FMK according to the manufacturer’s protocol
(CaspaTag Fluorescein Caspase Activity Kit; Intergen, Purchase, NY) to stain
activated caspases 1 through 9. After the cells were counterstained with PI,
they were analyzed by a FACS Calibur instrument (Becton Dickinson).
Synthesis of Duplex DNA. The synthesis of modified oligodeoxyribonucleotides containing dGS was accomplished by standard phosphoramidite
chemical methods with S6-DNP-dG-CE phosphoramidite (13). Oligodeoxyribonucleotides containing ara-C and 5FdU were synthesized and purified by
TriLink Biotechnologies (San Diego, CA). The 5⬘ ends of the single-stranded
oligodeoxyribonucleotides were radiolabeled by using the RTS T4 Kinase
Labeling System (Life Technologies, Inc., Gaithersburg, MD). Sequences of
the oligodeoxyribonucleotide duplexes are shown in Table 1. Oligodeoxyribonucleotide duplexes were prepared by annealing complementary single
strands and were purified by nondenaturing gel electrophoresis (12% polyacrylamide gel) at 4°C as previously described (13) or by Fast Protein Liquid
Chromatography using a Superdex 75 column (Amersham Pharmacia Biotech,
Piscataway, NJ).
Electrophoretic Mobility Shift Assay and Isolation of a Nucleoprotein
Complex. DNA binding assays were performed in a 20 –30-␮l solution containing buffer [20 mM Tris-HCl (pH 8.0); 50 mM NaCl; 3 mM MgCl2; 1 mM
DTT; 10% glycerol; 10 ng/␮l poly(deoxyinosinic-deoxycytidylic acid)] as
previously described (8). For the competition assay, 0 –225 nM of unlabeled
competitor were preincubated with nuclear extract (0 – 0.8 ␮g/␮l total protein)
in the presence of 2.5 mM ATP for 5 min at room temperature. Then,
P32-labeled oligo duplex was added to final concentration 10 nM, and the
reaction mixture was incubated for 15 min at room temperature. After addition
of loading buffer containing 40% sucrose and 0.04% bromphenol blue, the
samples were loaded onto 6% polyacrylamide gel and run for 2.5 h at 330 V
(⫹4°C). In a supershift assay, reaction mixture was prepared as described
above. After 10 min of incubation at room temperature, 5 ␮l (2.5 ␮g) of
anti-HMGB1 polyclonal antibody was added, and the reaction mixture was
incubated for additional 5 min before loading on the gel. For quantitative
measurement of bound oligonucleotide, the gels were dried and autoradiographed in a PhosphoroImager cassette overnight. The signal intensities were
normalized versus total radioactivity in the lane.
Radioactive fractions corresponding to the DNA-protein complex were
electroeluted from the gel and analyzed in a 7.5% SDS-polyacrylamide gel.
The bands containing the proteins of interest were excised from the gel, and the
proteins were digested with trypsin and subsequently identified by mass
spectroscopy, which was performed by Protana (Odense, Denmark).
Immunological Methods. Polyclonal rabbit antibodies against human
HMGB1 and HMGB2 (PharMingen, San Diego, CA) were used at a dilution
of 1:5000 and 1:200, respectively, for Western blot analysis. Polyclonal rabbit
antibody against the keyhole limpet hemocyanin-coupled peptide 491–505 of
ERp60 (SwissProt no. P30101) was raised and affinity-purified by Rockland,
Inc. (Gilbertsville, PA), and used at a 1:5000 dilution. Polyclonal goat antiHSC70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a
1:100 dilution. Monoclonal antibody to GAPDH (MAB 374; Chemicon,
Temecula, CA) was used at a dilution of 1:500. Secondary horseradish peroxidase-conjugated antibodies were used at dilutions ranging from 1:5000 to
1:10,000. The ECL-Plus Western blotting detection system (Amersham Pharmacia Biotech) was used to detect antibodies bound to the Western blots.
EMSA-Western Blot Analysis. After completion of EMSA electrophoresis in nondenaturing conditions as described in a preceding section, the
proteins were transferred to a Hybond-P membrane (Amersham Pharmacia
Biotech) under which a sheet of DE-81 paper (Whatman, Maidstone, England)
was placed, as described previously (20). After electrotransfer, the membrane
was incubated with the antibodies to the proteins that comprised the dGSDNA-indexing complex. Bound antibodies were detected by using secondary
antibodies and the ECL-Plus kit (Amersham Pharmacia Biotech). The radiolabeled DNA bound to the DE-8l paper was detected by exposing the dried
paper in a PhosphorImager cassette. For Western analysis of proteins in
supershift experiments, radioactive bands were excised, incubated at 70°C for
Table 1 Sequences of 34-mer synthetic DNA duplexes containing a single modified nucleoside
Oligonucleotide
duplex
a
Abbreviation
1
GC duplexa
2
GSC duplex
3
GST duplex
4
GaraC duplex
5
AfU duplex
Sequence
5⬘-ACTCTTGCCTTTAAGGAAAGTATCTAAATGCTTC
3⬘-TGAGAACGGAAATTCCTTTCATAGATTTACGAAG
5⬘-ACTCTTGCCTTTAAGGSAAAGTATCTAAATGCTTC
3⬘-TGAGAACGGAAATTCCTTTCATAGATTTACGAAG
5⬘-ACTCTTGCCTTTAAGGSAAAGTATCTAAATGCTTC
3⬘-TGAGAACGGAAATTCT TTTCATAGATTTACGAAG
5⬘-ACTCTTGCCTTTAAG GAAAGTATCTAAATGCTTC
3⬘-TGAGAACGGAAATTCaraCTTTCATAGATTTACGAAG
5⬘-ACTCTTGCCTTTAAGG AAAGTATCTAAATGCTTC
3⬘-TGAGAACGGAAATTCCfUTTCATAGATTTACGAAG
Control nonmodified DNA duplex.
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30 min in NuPage LDS Sample buffer (Invitrogen, Carlsbad, CA), and the
supernatant was loaded on the SDS-containing gel.
Immunoprecipitation. The anti-HMGB1 antibody (300 ␮g) was crosslinked to protein G conjugated to Sepharose (Pierce, Rockford, IL). This
complex was used with the Seize X Mammalian Immunoprecipitation kit
(Pierce) to immunoprecipitate cellular or nuclear proteins from Nalm6 cell
(50 mg of cells). Immobilization of antibody, loading of the cell lysate, and
washing was performed according to manufacturer’s instructions. Eluted proteins were further separated using 4 –12% NuPage Bis-Tris gel in MES buffer
(Invitrogen). After electrophoresis, the proteins were transferred to a PVDF
membrane for Western blot analysis.
MTT Cytotoxicity Assays. Fibroblast cell lines deficient and proficient in
Hmgb1 expression were generated as described previously (21). Thiopurine
cytotoxicity was determined using the MTT assay after incubation of C1
(Hmgb1⫺/⫺) and VA1 (Hmgb1⫹/⫹) cells with MP or vincristine for 3– 6 days
(22). Thiopurines were dissolved in DMEM without fetal bovine serum, and
the drug concentration was determined spectrophotometrically, as described
earlier (8). The IC50s were obtained by fitting a sigmoid Emax model to the
cell viability versus drug concentration data, determined in triplicate.
RESULTS
S-Phase Arrest and Apoptosis of MP-treated Synchronized
Cells. Untreated (control) synchronized Nalm6 cells progressed
through the first and second cell cycles after release from thymidine
block (Fig. 1A) and were no longer synchronized by the end of the
second cell cycle. When Nalm6 cells were treated with 10 ␮M MP
Fig. 2. Response of synchronized Nalm6 cells to MP treatment. Two h after the block
release, MP was added to a final concentration of 10 ␮M. The time at which MP was added
corresponds to the zero time point on the chart. E represents untreated (control) synchronized cells; 䡺 and f represent synchronized cells treated with 10 ␮M MP. A, fraction of
cells in the S phase of the cell cycle at each time point. B, relative number of cells that
incorporated BrdUrd into their DNA after the thymidine block was removed. Symbols are
same as in A. C, accumulation of dGS (expressed as dGS/100 dG residues) in DNA during
the first and the second cycles of division of synchronized Nalm6 cells. Data were
collected from three independent experiments (mean ⫾ SE).
Fig. 1. A, FACS analysis of DNA content in Nalm6 cells synchronized by a thymidine
block and released by medium change. Two h after the change, MP was added to a final
concentration of 10 ␮M. The time of MP addition corresponds to the zero time point. Cells
are shown without MP treatment (⫺MP, control) and after MP treatment (⫹MP) at 0, 4,
18, and at 34 h. B, fraction of cells containing DNA strand breaks (apoptotic cells), as
detected by the TUNEL assay. 䡺 depicts controls (no MP treatment); f, after MP
treatment. C, fraction of synchronized cells that contain activated caspases after treatment
with MP, as determined by flow cytometry using FAM-VAD-FMK as the substrate (see
“Materials and Methods”). 䡺, control (no MP treatment); f, after MP treatment. Data
were collected from three independent experiments (mean ⫾ SE).
(IC50 ⫽ 3.7 ⫾ 0.8 ␮M after 48 h), 2 h after the block was released,
cells progressed through the cell cycle during the first 18 h after the
addition of MP (Fig. 1A, 18 h). After beginning the second cell cycle,
additional progression of treated cells ceased, and arrest occurred
during S phase (Fig. 1A, 34 h).
TUNEL assay revealed that up to 12% of synchronized MP-treated
cells accumulated DNA strand breaks after 34 h of incubation (i.e., in
the second cycle) with MP (Fig. 1B). Consistent with this finding,
FACScan analysis demonstrated activation of caspases in ⬃20% of
MP-treated cells, compared with 4% in untreated cells (Fig. 1C).
Caspase activation occurred simultaneously with accumulation of
cells in S phase and the increase of sub-G1 fraction (up to 14 –17%)
in synchronized Nalm6 cells treated with MP. Interestingly, both
accumulation of DNA strand breaks and caspase activation took place
only after treated cells entered S phase of the second cycle (Figs. 1,
A–C and 2A).
Continuation of DNA Synthesis in Synchronized Nalm6 Cells
Treated with MP. Pulse-chase experiments showed that the level of
BrdUrd incorporation into DNA was similar in untreated and MPtreated cells, indicating that DNA synthesis was not abrogated by MP
treatment (Fig. 2, A and B). dGS incorporation (0.06%, about one
dGS/1700 dG) was detected in MP-treated cells after completion of
the first cycle and increased to 0.4% during the second cycle (one
dGS/250 dG; Fig. 2C), consistent with our previous findings in unsynchronized cells (13). The level of dGS incorporated into DNA
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increased during the second cycle of cell division in parallel with the
relative number of cells in S-phase, also showing that DNA synthesis
was not abrogated (Fig. 2, A–C).
High Affinity of Nuclear Proteins to dGS-DNA Duplex. High
affinity of nuclear extract proteins from mismatch-deficient lymphoblastic cells to dGS-DNA was demonstrated by EMSA experiments
with 34-mer oligo duplexes. Titration of the complex with a competitor oligo duplex that had the identical sequence, except contained
only natural nucleosides, demonstrated stability of the complex in the
presence of 4 –5-fold excess of competitor (Fig. 3, A and B). Further
addition of competitor resulted in a decrease of the radiolabeled
complex because of displacement of the labeled dGS-DNA duplex
from the complex with nonlabeled competitor. Therefore, subsequent
EMSA experiments were performed in the presence of 5-fold excess
of nonradioactive competitor. An increase of the protein concentration
in the reaction mix led to a linear increase of band intensity, further
confirming the specificity of complex formation (Fig. 3, C and D).
Identification and Characterization of a dGS-DNA-Protein
Complex. A dGS-DNA-protein complex, formed in the presence of
radiolabeled 34-mer DNA duplexes (see Table 1) containing one
modified base, was isolated from Nalm6 nuclear extract by EMSA
electrophoresis (Fig. 4A). Analysis by SDS-PAGE revealed that the
complex contained four or five major proteins (Fig. 4B). Mass spectroscopy established that the protein with a molecular mass of 70 kDa
was human HSC70 (SwissProt no. P11142; total coverage: 389/646
amino acids, 60%; average mass, 71,126); the protein with a molecular mass of 58 kDa was the putative ERp60 (SwissProt no. P30101;
total coverage: 225/505 amino acids, 45%; average mass, 57,182); the
protein with molecular mass of 25 kDa was human HMG protein 1
HMGB1 (SwissProt no. P09429; total coverage: 109/214 amino acids,
51%; average mass, 24,933); the protein with a molecular mass of 23
kDa was human HMGB2 (SwissProt no. P26583; total coverage:
145/208 amino acids, 70%; average mass 24,073); and the protein
Fig. 4. Identification of the nuclear proteins from Nalm6 cells involved in binding of
dGS-DNA. A, the DNA-protein complex binding dGS-modified DNA (indicated by
arrow) was isolated by EMSA (Lane 1). Incubation of the complex with anti-HMGB1
antibody resulted in formation of the supershift band (Lane 2). B, SDS-PAGE analysis of
proteins eluted from the DNA-protein complex; proteins were visualized by silver staining
(42). The most intense bands were excised, and the proteins were identified by mass
spectroscopy. C, Western blot analysis of proteins extracted from the DNA-protein
complex band (A, Lane 1) and the supershift band (A, Lane 2, top band). Extracted
proteins were loaded on three separate membranes and developed with anti-GAPDH Ab,
anti-HSC70 Ab, anti-HMGB2 Ab, as indicated on the membrane images. Proteins
extracted from DNA-protein complex (A, Lane 1) are in Lane 1, and proteins from the
supershift band (A, Lane 2, top band) are in Lane 2.
Fig. 3. Preferential binding of Nalm6 nuclear extract proteins with dGS-DNA oligo
duplex. A, EMSA experiments were performed with 10 nM duplex 2 (see Table 1) with
increasing concentration of duplex 1 (0 –225 nM). B, quantification of DNA-protein
binding shown on A. The results present three independent experiments (mean ⫾ SE). C,
EMSA experiments were performed with 10 nM duplex 2 in the presence of 50 nM duplex
1 with increasing concentrations of nuclear proteins (0 – 0.8 ␮g/␮l). D, quantification of
DNA-protein binding shown on C. The results present three independent experiments
(mean ⫾ SE).
with a molecular mass of 37 kDa was GAPDH (SwissProt no.
P04406; total coverage: 77/334 amino acids, 23%; average mass,
36,093). Identification of GAPDH as a component of the complex
corroborates our earlier findings obtained by NH2-terminal sequencing (8).
Additional confirmation of the identity of the proteins was provided
by the results of EMSA in the presence of antibodies specific to
identified proteins. Two of the antibodies gave a notable effect:
anti-HMGB1 antibody resulted in a supershift of the dGS-DNAprotein complex (Fig. 4A, Lanes 1 and 2); and anti-GAPDH antibody
abrogated formation of the complex, as reported earlier (8). Treatment
with antibodies to HMGB2, ERp60, and HSC70 did not change
mobility of the complex in EMSA.
In a separate set of EMSA experiments, the radioactive band
formed after the treatment of the reaction mixture with anti-HMGB1
antibody (Fig. 4A, Lane 2, top band) was excised and subjected to
Western blot analysis. The presence of HMGB2, GAPDH, and
HSC70 proteins in the excised (supershift) band was confirmed based
on immunoreactivity to the antibodies and molecular weights of the
proteins, as depicted in Fig. 4C. We were unable to unambiguously
identify ERp60 in this band because of cross-reactivity of the secondary antibody to anti-HMGB1 antibody and anti-ERp60 antibody.
Identities of the major components of the dGS-DNA-protein complex
(HMGB1, HMGB2, HSC70, ERp60, GAPDH) were also confirmed
by Western analysis after the transfer of the proteins from the EMSA
gel onto a PVDF membrane, with the radiolabeled oligonucleotide
duplex retained on the ion-exchange paper underlying the PVDF
membrane (data not shown).
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Coimmunoprecipitation experiments were performed using antiHMGB1 antibody. HMGB1 was isolated from the nuclear extract of
untreated Nalm6 cells by affinity chromatography using anti-HMGB1
antibody immobilized on protein G-conjugated Sepharose. Western
blot analysis of the bound proteins showed that three components of
the complex (i.e., HMGB2, ERp60, HSC70) coimmunoprecipitated
with HMGB1 (Fig. 5, Lanes 2–5). We did not detect GAPDH in the
coimmunoprecipitated fraction.
Incubation of the nuclear extract with synthetic duplexes containing
the modified nucleosides ara-C or 5FdU (Table 1) resulted in protein
binding similar to that observed when the extract was incubated with
dGS-containing DNA duplexes (Fig. 6A, Lanes 7 and 9). Again,
addition of anti-HMGB1 antibody to the reaction mixture resulted in
supershift of the complex, suggesting that the same complex was
involved in the binding of dGS-, araC-, and 5FdU-containing oligonucleotide duplexes (Fig. 6A, Lanes 8 and 10). Comparison of band
intensities indicated that the protein complex has ⬃4 –5-fold greater
affinity for chemically modified oligonucleotide duplexes than for
duplex DNA containing only natural nucleosides (Fig. 6B).
Thiopurine Resistance of Hmgb1-deficient Murine Fibroblasts.
Immortalized fibroblast cell lines established from Hmgb1⫺/⫺ and
wild-type (Hmgb1⫹/⫹) murine embryos (21) were used to assess
thiopurine sensitivity by MTT assay. Estimation of cell viability in C1
(Hmgb1⫺/⫺) and VA1 (Hmgb1⫹/⫹) cells after MP treatment revealed
that after 5 days of incubation, Hmgb1⫺/⫺ cells were ⬃10-fold more
resistant to MP treatment, compared with Hmgb1⫹/⫹ fibroblasts
(IC50C1 ⫽ 8.9 ⫾ 0.7 ␮M and IC50VA1 ⫽ 0.78 ⫾ 0.11 ␮M, P ⫽ 0.0001;
Fig. 7). In contrast, C1 (Hmgb1⫺/⫺) and VA1 (Hmgb1⫹/⫹) cells
demonstrated similar viability after 5 days of incubation with vincristine (IC50C1 ⫽ 0.28 ⫾ 0.032 ␮M; IC50VA1 ⫽ 0.39 ⫾ 0.094 ␮M), a
cytotoxic antileukemic agent that is not incorporated into DNA.
DISCUSSION
The thiopurine drugs MP, thioguanine, ␣-2⬘-deoxythioguanosine,
and ␤-2⬘-deoxythioguanosine form a tight cluster when grouped according to their activity or gene expression profiles in 60 sensitive and
resistant human cancer cell lines, implying that thiopurines have a
similar molecular mechanism of cytotoxicity (11). Although the
MMR system was shown to be an important determinant of thiopurine
Fig. 6. DNA-protein complex formed in the presence of DNA containing nonnatural
nucleosides. A, EMSA experiment was performed in the presence of 10 nM 5⬘-[32P]labeled synthetic DNA duplexes 2– 4 (see Table 1) that differed by the central nucleosides
involved in bp formation: GSC in duplex 2 (Lanes 1 and 2), GST in duplex 3 (Lanes 3 and
4), GC in duplex 1 (Lanes 5 and 6), GaraC in duplex 4 (Lanes 7 and 8), or A5FU in duplex
5 (Lanes 9 and 10). Five-fold excess of nonradiolabelled duplex 1 was added to each
reaction mixture. Anti-HMGB1 antibody was added to one reaction in each pair (Lanes 2,
4, 6, 8, and 10), as indicated. B, quantification of the DNA-protein complexes (indicated
by arrow) formed with chemically modified oligonucleotide duplexes 1–5 (see Table 1).
Fig. 7. The absence of Hmgb1 alters sensitivity to MP. Viability of Hmgb1⫺/⫺ (C1)
and Hmgb1⫹/⫹ (VA1) cells (MEFs) after 5 days of MP treatment, determined by MTT
assay. 䉫, C1 cells; F, VA1 cells. Each point represents the results of three parallel
experiments (mean ⫾ SE). In contrast, Hmgb1⫺/⫺ cells were not more resistant to
vincristine (see “Results”).
Fig. 5. HMGB1-associated proteins coimmunoprecipitated by the anti-HMGB1 antibody from nuclear extracts of untreated Nalm6 cells. Lane 1 depicts proteins after silver
staining. Lanes 2–5 depict results of Western analysis using the anti-HMGB1 antibody,
anti-HMGB2 antibody, anti-ERp60 antibody, and anti-HSC70 antibody, as indicated.
sensitivity in several types of cells (17), our prior studies have shown
that MMR-deficient human leukemia cells can be highly sensitive to
thiopurine treatment (8, 13). This observation led to our current
studies to elucidate alternative mechanisms by which cells respond to
the DNA modification after thiopurine treatment. To this end, we used
nuclear extracts from human leukemia cells (i.e., Nalm6) that are
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MULTIPROTEIN SENSOR OF CHEMICALLY MODIFIED DNA
highly sensitive to thiopurines (IC50 ⫽ 3.7 ⫾ 0.8 ␮M after 48 h
exposure to MP) but do not have detectable levels of MSH2 or MSH6,
essential components of the MMR system (8).
Treated cells progressed successfully through G2-M of the first cell
cycle and G1 phase of the second cell cycle, without cycle arrest (Fig.
1). During the second cell cycle, additional dGS was incorporated into
DNA, a result consistent with formation and accumulation of deoxythioguanosine triphosphate within cells (23) and with continued DNA
synthesis. As Fig. 1 and 2, A and B, depict, thiopurine metabolites
neither inhibit DNA replication nor activate checkpoints that determine entrance into S phase. Moreover, DNA synthesis was not abrogated, as evidenced by the incorporation of BrdUrd and dGS into
DNA (Fig. 2, B and C). However, thiopurine-treated cells did not
progress beyond the second S phase and did not accomplish duplication of DNA (Fig. 1, 34 h ⫹MP).
Interestingly, apoptotic changes in MP-treated cells became prominent only during the second S phase, after incorporation of dGS into
DNA. Both activation of caspases and accumulation of DNA breaks
occurred, indicating activation of the apoptotic cascade in the second
cell cycle (Fig. 1, B and C). These results are consistent with earlier
findings in nonhuman CHO and L1210 cell lines, which demonstrated
accumulation of DNA breaks in the second cycle after thiopurine
treatment (24, 25). The fact that initiation of apoptosis occurs in the
second S phase after the incorporation of dGS into DNA suggests
existence of cycle-specific mechanism(s) that are sensitive to such
DNA modification.
On the basis of the above findings in MMR-deficient cells, we
hypothesized that chemically modified DNA with dGS-inserts must be
a substrate for a protein(s) that recognizes DNA distortion rather than
formation of specific mispairs. Formation of DNA-protein complex
with such altered DNA may be considered as the initial step toward
activation of DNA repair or apoptosis (26 –28). Structural analysis of
dGS-containing duplex DNA has demonstrated local changes in geometry and dynamics of the double helix around the site of dGS
incorporation (12), without loss of structural integrity of the DNA
duplex. Thus, the protein(s) recognizing dGS-modified DNA is detecting relatively modest and localized changes in DNA structure.
Synthetic DNA duplexes containing nonnatural nucleosides provide a useful tool to characterize cellular components sensitive to
DNA modification (29). We used a similar approach to identify
cellular components sensitive to chemical modifications that do not
affect integrity of DNA. In contrast to incorporation of 2⬘, 2⬘-difluorodeoxycitidine into DNA (29), dGS does not cause cessation of DNA
strand elongation. Therefore, to characterize and isolate the protein
complex interacting with dGS-modified DNA, we performed EMSA
using synthetic oligonucleotide duplexes containing thioguanine basepaired with cytosine or thymine in the central position of the duplex
(Table 1). Titration experiments demonstrated that in the presence of
4 –5-fold excess of the nonmodified oligo duplex, dGS-DNA remained
bound with the identified nuclear complex, demonstrating higher
affinity of these nuclear proteins to modified DNA (Fig. 3, A and B).
Selectivity for modified DNA is also demonstrated by preferential
complex formation with oligo duplexes containing nonnatural nucleoside inserts (Fig. 6).
In this study, we demonstrated that the nuclear complex, which
recognizes nonnatural nucleosides incorporated into DNA, contains
HMGB1, HMGB2, HSC70, ERp60, and GAPDH proteins and binds
dGS-modified DNA with substantially (4 –5-fold) higher affinity than
DNA containing only natural nucleotides. Coimmunoprecipitation of
HMGB1 with three components of the complex from nuclear extracts
of untreated cells (Fig. 5) indicates that these proteins interact in the
absence of drug challenge, rather than assemble subsequent to DNA
modification. Moreover, a similar DNA-protein complex is formed in
the presence of oligonucleotide duplexes modified by incorporation of
two other chemically distinct nonnatural nucleosides, araC and 5FdU
(Table 1), as evidenced by supershift experiments in the presence of
polyclonal rabbit anti-HMGB1 antibody (Fig. 6). Thus, three nonnatural nucleoside analogs (dGS, araC, and 5FdU) create DNA lesions
that are recognized by this complex, indicating that this multiprotein
complex may be a general sensor of DNA that has been modified by
incorporation of structurally distinct nonnatural nucleosides.
HMGB1 and HMGB2 are abundant proteins localized in the nucleus (30, 31), and ERp60, a member of the large protein disulfide
isomerase family, is localized in the nucleus and cytosol (32). Two
components of the complex, a multifunctional protein GAPDH, (33)
and HSC70, which is associated with the nuclear matrix (34), shuttle
into the nucleus after stress, e.g., thiopurine treatment (8). All of the
identified proteins in the complex have protein-protein binding properties and participate in the formation of homo- or heterocomplexes,
and four of five proteins (HMGB1/2, ERp60, and GAPDH) have
known DNA-binding properties (30, 32, 33, 35). Among the components of this complex, it is most likely that HMGB1 and HMGB2 are
involved in direct binding with modified DNA. Although these proteins do not reveal sequence specificity, they have increased affinity
to cruciform DNA, B-Z DNA junction (30) and DNA modified with
cisplatin, an anticancer agent that significantly changes DNA geometry (36, 37). HMGB1/2 proteins contain two similar tandem HMGbox domains A and B, organized in a characteristic L-shape consisting
of three ␣-helices and are capable of introducing bends into DNA
(30). It is plausible that dGS incorporation facilitates the ability of
DNA to acquire a bent conformation when bound to HMGB1/2
proteins. Because destabilizing alterations within the structure decrease DNA rigidity (38), we speculate that HMGB1/2 binding to
flexible regions in DNA could provide a common mechanism for
active scanning of DNA damage. HMG proteins are considered architectural regulators of transcription; therefore, DNA modifications
that increase DNA binding of HMGB1/2 may initiate a stress response
through transcriptional activation. Also, HMG functional motifs are
hypothesized to play a major role in facilitating the orderly progression of many DNA-related activities, including replication, recombination, and repair (31).
To substantiate the hypothesis about involvement of HMGB1 into
DNA stress response, we determined thiopurine sensitivity in immortalized fibroblast cell lines derived from Hmgb1-deficient and
-proficient murine embryos (21). Fibroblasts lacking Hmgb1 were
⬃10-fold more resistant (higher IC50) to MP (Fig. 7) but demonstrated
similar sensitivity to vincristine, an antileukemic agent that is cytotoxic by arresting cell mitosis rather than incorporation into DNA
(39). The direct interaction of HMGB1-containing protein complex
with dGS-DNA, coupled with the lower sensitivity in Hmgb1⫺/⫺ cells
suggests that the HMGB1-containing complex plays an important role
in cellular response to chemical modification of DNA. Recently,
HMGB1 was shown to associate with the nuclei in apoptotic cells,
indicating that during apoptosis, chromatin undergoes some chemical
or structural transition that makes it susceptible to HMGB1 binding
(40). Elucidation of the details of HMGB1 interactions with its various partners has considerable therapeutic potential (41). Investigation
of the interactions between HMGB1, HMGB2, and DNA containing
different nonnucleoside analogs, as well as the interactions between
HMGB1 and its protein partners (HMGB2, HSC70, Erp60 and
GAPDH), is ongoing in our lab.
Identification of a new multiprotein nuclear complex and its role in
cell sensitivity to thiopurines in MMR-deficient cells opens new
insights to a heretofore unrecognized cellular response to incorporation of nonnatural nucleosides into DNA. These findings provide the
foundation for future studies to elucidate the function of individual
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MULTIPROTEIN SENSOR OF CHEMICALLY MODIFIED DNA
components of this complex and their role in determining the sensitivity of cells to anticancer agents that exert their effects via incorporation into DNA. An important future direction will be to determine
whether this multiprotein complex is the initial sensor of DNA modification that provides the primary pro-apoptotic signal in response to
incorporation of nonnatural nucleosides in DNA.
ACKNOWLEDGMENTS
We thank Dr. R. A. Ashmun and the staff of the Flow Cytometry and Cell
Sorting Shared Resource at St. Jude Children’s Research Hospital for their
invaluable help and expertise, Dr. J. C. Panetta for assistance with statistical
analysis of the results, X. S. Jiang for help with experiments involving synchronized cells, and M. L. Hankins, M. V. Mane, and Y. Su for their excellent
technical assistance.
REFERENCES
1. Johnstone, R. W., Ruefli, A. A., and Lowe, S. W. Apoptosis: a link between cancer
genetics and chemotherapy. Cell, 108: 153–164, 2002.
2. Huang, P., and Plunkett, W. Fludarabine- and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event. Cancer Chemother. Pharmacol., 36:
181–188, 1995.
3. Miyashita, T., and Reed, J. C. Bcl-2 oncoprotein blocks chemotherapy-induced
apoptosis in a human leukemia cell line. Blood, 81: 151–157, 1993.
4. Kaufmann, S. H., and Earnshaw, W. C. Induction of apoptosis by cancer chemotherapy. Exp. Cell Res., 256: 42– 49, 2000.
5. Nelms, B. E., Maser, R. S., MacKay, J. F., Lagally, M. G., and Petrini, J. H. In situ
visualization of DNA double-strand break repair in human fibroblasts. Science
(Wash. DC), 280: 590 –592, 1998.
6. Paull, T. T., Cortez, D., Bowers, B., Elledge, S. J., and Gellert, M. From the cover:
direct DNA binding by Brca1. Proc. Natl. Acad. Sci. USA, 98: 6086 – 6091, 2001.
7. Griffin, S., Branch, P., Xu, Y. Z., and Karran, P. DNA mismatch binding and incision
at modified guanine bases by extracts of mammalian cells: implications for tolerance
to DNA methylation damage. Biochemistry, 33: 4787– 4793, 1994.
8. Krynetski, E. Y., Krynetskaia, N. F., Gallo, A. E., Murti, K. G., and Evans, W. E. A
novel protein complex distinct from mismatch repair binds thioguanylated DNA. Mol.
Pharmacol., 59: 367–374, 2001.
9. Elion, G. B. The purine path to chemotherapy. Science (Wash. DC), 244: 41– 47,
1989.
10. LePage, G. A. Basic biochemical effects and mechanism of action of 6-thioguanine.
Cancer Res., 23: 1202–1206, 1963.
11. Scherf, U., Ross, D. T., Waltham, M., Smith, L. H., Lee, J. K., Tanabe, L., Kohn,
K. W., Reinhold, W. C., Myers, T. G., Andrews, D. T., Scudiero, D. A., Eisen, M. B.,
Sausville, E. A., Pommier, Y., Botstein, D., Brown, P. O., and Weinstein, J. N. A gene
expression database for the molecular pharmacology of cancer [see comments]. Nat.
Genet., 24: 236 –244, 2000.
12. Somerville, L., Krynetski, E. Y., Krynetskaia, N. F., Beger, R. D., Zhang, W.,
Marhefka, C. A., Evans, W. E., and Kriwacki, R. W. Structure and dynamics of
thioguanine-modified duplex DNA. J. Biol. Chem., 27B, 2003.
13. Krynetskaia, N. F., Krynetski, E. Y., and Evans, W. E. Human RNase H-mediated
RNA cleavage from DNA-RNA duplexes is inhibited by 6-deoxythioguanosine
incorporation into DNA. Mol. Pharmacol., 56: 841– 848, 1999.
14. Krynetskaia, N. F., Cai, X., Nitiss, J. L., Krynetski, E. Y., and Relling, M. V.
Thioguanine substitution alters DNA cleavage mediated by topoisomerase II. FASEB
J., 14: 2339 –2344, 2000.
15. Relling, M. V., Yanishevski, Y., Nemec, J., Evans, W. E., Boyett, J. M., Behm, F. G.,
and Pui, C. H. Etoposide and antimetabolite pharmacology in patients who develop
secondary acute myeloid leukemia. Leukemia (Baltimore), 12: 346 –352, 1998.
16. Relling, M. V., Rubnitz, J. E., Rivera, G. K., Boyett, J. M., Hancock, M. L., Felix,
C. A., Kun, L. E., Walter, A. W., Evans, W. E., and Pui, C. H. High incidence of
secondary brain tumours after radiotherapy and antimetabolites [see comments].
Lancet, 354: 34 –39, 1999.
17. Karran, P. Mechanisms of tolerance to DNA damaging therapeutic drugs. Carcinogenesis (Lond.), 22: 1931–1937, 2001.
18. Pichierri, P., Franchitto, A., Piergentili, R., Colussi, C., and Palitti, F. Hypersensitivity
to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein
in recombinational repair. Carcinogenesis (Lond.), 22: 1781–1787, 2001.
19. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. Accurate transcription initiation by
RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic
Acids Res., 11: 1475–1489, 1983.
20. Bianchi, M. E., Beltrame, M., and Paonessa, G. Specific recognition of cruciform
DNA by nuclear protein HMG1. Science (Wash. DC), 243: 1056 –1059, 1989.
21. Calogero, S., Grassi, F., Aguzzi, A., Voigtlander, T., Ferrier, P., Ferrari, S., and
Bianchi, M. E. The lack of chromosomal protein Hmg1 does not disrupt cell growth
but causes lethal hypoglycaemia in newborn mice. Nat. Genet., 22: 276 –280, 1999.
22. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. Methods, 65: 55– 63, 1983.
23. Krynetski, E. Y., and Evans, W. E. Mercaptopurine and related compounds. In: H. P.
Lipp (ed.), Anticancer Drug Toxicity, pp. 392– 429. New York: Marcel Dekker, Inc.,
1999.
24. Fairchild, C. R., Maybaum, J., and Kennedy, K. A. Concurrent unilateral chromatid
damage and DNA strand breakage in response to 6-thioguanine treatment. Biochem.
Pharmacol., 35: 3533–3541, 1986.
25. Christie, N. T., Drake, S., Meyn, R., and Nelson, J. A. 6-thioguanine-induced DNA
damage as a determinant of cytotoxicity in cultured Chinese hamster ovary cells.
Cancer Res., 44: 3665–3671, 1984.
26. da Silva, C. P., de Oliveira, C. R., da Conceicao, M., and de Lima, P. Apoptosis as
a mechanism of cell death induced by different chemotherapeutic drugs in human
leukemic T-lymphocytes. Biochem. Pharmacol., 51: 1331–1340, 1996.
27. Hortelano, S., and Bosca, L. 6-Mercaptopurine decreases the Bcl-2/Bax ratio and
induces apoptosis in activated splenic B lymphocytes. Mol. Pharmacol., 51: 414 –
421, 1997.
28. Tsurusawa, M., Saeki, K., and Fujimoto, T. Differential induction of apoptosis on
human lymphoblastic leukemia Nalm-6 and Molt-4 cells by various antitumor drugs.
Int. J. Hematol., 66: 79 – 88, 1997.
29. Achanta, G., Pelicano, H., Feng, L., Plunkett, W., and Huang, P. Interaction of p53
and DNA-PK in response to nucleoside analogues: potential role as a sensor complex
for DNA damage. Cancer Res., 61: 8723– 8729, 2001.
30. Bustin, M., and Reeves, R. High-mobility-group chromosomal proteins: architectural
components that facilitate chromatin function. Prog. Nucleic Acid Res. Mol. Biol.,
54: 35–100, 1996.
31. Bustin, M. Regulation of DNA-dependent activities by the functional motifs of the
high-mobility-group chromosomal proteins. Mol. Cell. Biol., 19: 5237–5246, 1999.
32. Ferraro, A., Altieri, F., Coppari, S., Eufemi, M., Chichiarelli, S., and Turano, C.
Binding of the protein disulfide isomerase isoform ERp60 to the nuclear matrixassociated regions of DNA. J. Cell Biochem., 72: 528 –539, 1999.
33. Sirover, M. A. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim. Biophys. Acta, 1432:
159 –184, 1999.
34. Jin, M. L., Zhang, P., Ding, M. X., Yun, J. P., Chen, P. F., Chen, Y. H., and Chew,
Y. Q. Altered expression of nuclear matrix proteins in etoposide-induced apoptosis in
HL-60 cells. Cell Res., 11: 125–134, 2001.
35. Xanthoudakis, S., and Nicholson, D. W. Heat-shock proteins as death determinants.
Nat. Cell Biol., 2: E163–E165, 2000.
36. Cohen, S. M., and Lippard, S. J. Cisplatin: from DNA damage to cancer chemotherapy. Prog. Nucleic Acid Res. Mol. Biol., 67: 93–130, 2001.
37. Pasheva, E. A., Ugrinova, I., Spassovska, N. C., and Pashev, I. G. The binding affinity
of HMG1 protein to DNA modified by cisplatin and its analogs correlates with their
antitumor activity. Int. J. Biochem. Cell Biol., 34: 87–92, 2002.
38. Marathias, V. M., Jerkovic, B., and Bolton, P. H. Damage increases the flexibility of
duplex DNA. Nucleic Acids Res., 27: 1854 –1858, 1999.
39. Lipp, H. P., and Bokemeyer, C. Clinical pharmacokinetics of cytostatic drugs:
efficacy and toxicity. In: H. P. Lipp (ed.), Anticancer Drug Toxicity, pp. 11–201.
New York: Marcel Dekker, Inc., 1999.
40. Scaffidi, P., Misteli, T., and Bianchi, M. E. Release of chromatin protein HMGB1 by
necrotic cells triggers inflammation. Nature (Lond.), 418: 191–195, 2002.
41. Bustin, M. At the crossroads of necrosis and apoptosis: signaling to multiple cellular
targets by HMGB1. Sci STKE., 2002: PE39, 2002.
42. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. Mass spectrometric sequencing
of proteins from silver-stained polyacrylamide gels. Anal. Chem., 68: 850 – 858,
1996.
106
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Research.
A Nuclear Protein Complex Containing High Mobility Group
Proteins B1 and B2, Heat Shock Cognate Protein 70, ERp60,
and Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved
in the Cytotoxic Response to DNA Modified by Incorporation
of Anticancer Nucleoside Analogues
Eugene Y. Krynetski, Natalia F. Krynetskaia, Marco E. Bianchi, et al.
Cancer Res 2003;63:100-106.
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