Steroid hormones induce HMG1 overexpression and
sensitize breast cancer cells to cisplatin
and carboplatin
Qing He, Cynthia H. Liang, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by Stephen J. Lippard, March 13, 2000
Cisplatin is an anticancer drug that has enjoyed remarkable success
against testicular tumors, but dose limiting side-effects have limited
its application against a broader range of cancers. Previous
studies have shown that high-mobility group (HMG) domain proteins
such as HMG1 sensitize cells to cisplatin by shielding its major
DNA adducts from nucleotide excision repair. Estrogen treatment
increases HMG1 mRNA levels in breast cancer MCF-7 cells. Herein,
we describe that treatment of human cancer cells having steroid
hormone receptors with the appropriate hormone, estrogen
and͞or progesterone, significantly increases the potency of cisplatin
and its analogue carboplatin by causing the overexpression of
HMG1. These findings suggest that the proper combination of
these drugs, which are already approved by the Food and Drug
Administration, could have potential benefit in treating tumors
such as ovarian or breast that carry the hormone receptors.
Cisplatin [cis-diamminedichloroplatinum(II) also known as
cis-DDP] is a widely used antitumor drug for the treatment
of testicular, breast, ovarian, lung, head, and neck tumors
(1–5). DNA is the primary cytotoxic target of cisplatin in vivo
(6). The major cisplatin-DNA adducts are 1,2-intrastrand
d(GpG) and d(ApG) crosslinks, and the adducts significantly
bend and distort normal B DNA (7, 8). This structural
distortion in DNA affects DNA replication and transcription
(9–13) and is recognized by a variety of structure-specific
DNA-binding proteins, such as DNA repair and high-mobility
group (HMG) domain proteins (13).
HMG domain proteins are architectural proteins that facilitate
cellular functions requiring chromosomal DNA (14). HMG
domain proteins bind specifically to the major cisplatin-DNA
adducts, forming a stable platinum-DNA-protein ternary complex
(15, 16). There is evidence implicating the involvement of
the platinum-DNA-protein complex in mediating cisplatin cytotoxicity
by blocking nucleotide excision repair of the DNA
damage, a process termed repair shielding. In yeast, interruption
of the HMG domain protein Ixr1 caused a 2- to 6-fold desensitization
to cisplatin compared with that in wild-type cells (17,
18). In vitro experiments revealed that a variety of HMG domain
proteins, including HMG1, tsHMG, and SRY, blocked removal
of cisplatin intrastrand d(GpG) adducts when added in a nucleotide
excision repair assay (19–21). Depleting HMG1 and
HMG2 from cell extracts by immunoprecipitation enhanced
excision repair of cisplatin-modified DNA (22). Furthermore,
introducing HMG2 by transfection enhanced the cisplatin sensitivity
of a lung adenocarcinoma cell line (23). Until the present
work, however, there was, to our knowledge, no evidence that
overexpression of an HMG domain protein via a natural signal
transduction pathway in human cancer cells could increase their
sensitivity to cisplatin.
HMG1 is a structure-specific HMG domain protein with little
or no sequence specificity. It is abundant in all tissues and species
(24). HMG1 binds preferentially to cruciform DNA, cisplatinmodified
DNA, and other distorted structures (14). It has a
multitude of functions including association with chromosomes
and interaction with Oct, Hox, p53 protein, and some components
of the basal transcriptional machinery (25–29). HMG1 also
functions as an architectural protein to facilitate the binding of
steroid hormone receptors, such as estrogen receptors (ER),
progesterone receptors (PR), androgen receptors, and glucocorticoid
receptors to their cognate DNA binding sites (30). Binding
of ER to estrogen-responsive elements and PR to progesteroneresponsive
elements induces DNA bending (31) and, accordingly,
increases HMG1 affinity. The binding of HMG1 further
alters the structure of the target DNA and facilitates formation
of a more stable receptor-DNA complex (32–34). The transcriptional
activity of these steroid hormones is enhanced in
mammalian cells that are expressing HMG1 transiently (30, 32).
Mice deficient in HMG1 die of hypoglycemia, implicating a
role for HMG1 in glucocorticoid-dependent gene regulatory
pathways (35).
The rationale for the discovery described herein was derived
from previous work reporting that the mRNA level of chromosomal
architectural protein HMG1 is up-regulated when human
MCF-7 breast cancer cells are treated with estrogen (36). We
corroborate such overexpression at the protein level. Because of
the inverse relationship between the levels of HMG domain
proteins and the ability of cells to repair cisplatin-DNA adducts
both in vivo and in vitro, described above, we predicted that
steroid hormone treatment would sensitize these cells to cisplatin.
The present results confirm this prediction and suggest that
a combination of cisplatin or carboplatin with estrogen and͞or
progesterone, all of which have been approved by the Food and
Drug Administration, may be of clinical significance.
Materials and Methods
Cell Culture. Cells were grown in DMEM (GIBCO͞BRL) containing
10% (vol͞vol) heat-inactivated FBS (GIBCO͞BRL), 2
mM glutamine, 100 units͞ml penicillin, and 100 ␮g͞ml streptomycin
at 37°C under a 5% CO2(g) atmosphere.
Clonogenic Assays. Cells were seeded on 6-well plates (Corning)
at a density of 400 cells per well. After 24 h, a fresh stock of
steroid hormone, estrogen or progesterone, was prepared in
N,N-dimethylformamide and added to the plates at a final
hormone concentration described below for each experiment.
Control plates were treated with the same volume of N,Ndimethylformamide
without hormone. For cotreatment, the
hormone was added at the same time as cisplatin. For pretreatment,
the hormone was added 2 h, 4 h, or 24 h before cisplatin.
After 4 h of cisplatin treatment, the cells were washed with PBS,
and fresh medium was added. After 10 days, the cell colonies
Abbreviations: ER, estrogen receptor; PR, progesterone receptor; trans-DDP, trans-
diamminedichloroplatinum(II).
*To whom reprint requests should be addressed. E-mail: lippard@lippard.mit.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.100108697.
Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.100108697
5768–5772 ͉ PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11
were stained with a 1% methylene blue (Fluka)͞50% (vol/vol)
ethanol solution and were then counted. Each point is an average
of three independent determinations Ϯ 1 SD (Eq. 1) of the cell
count.
SD ϭ ͱ͸͑Xi Ϫ X៮ ͒2
N Ϫ 1
[1]
Immunofluorescence. The cells were grown to 70% confluence on
12-mm glass coverslips in 6-well plates and subsequently treated
with estrogen or progesterone for 0–24 h. After hormone
treatment, the cells were permeabilized with 25% (vol͞vol)
acetic acid in methanol for 10 min at room temperature and
washed with PBS. The permeabilized cells were incubated for 30
min at 37°C with 1:100 dilution (for MCF-7 cells) or 1:500
dilution (for Evsa-T cells) of anti-HMG1 polyclonal antibody
(PharMingen), washed, and incubated subsequently with 1:200
dilution of goat anti-rabbit IgG conjugated to fluorescein (Biosource
International, Camarillo, CA) for 30 min at 37°C. Finally,
the cells were visualized with a fluorescent light microscope
(Zeiss Axiophot).
Flow Cytometry. About 106
cells were collected, fixed in 70%
(vol͞vol) ethanol, and then incubated in 1 ml of PBS containing
50 ␮g͞ml propidium iodide (Sigma) and 250 ␮g͞ml RNase A
(Roche Molecular Biochemicals) at 37°C for 30 min to stain the
DNA and eliminate RNA. For each sample, 100,000 cells were
analyzed in a fluorescence-activated cell sorter (FACScan, Becton
Dickinson) for the cell-cycle profile by using CELL QUEST and
MODIFIT software.
Analysis of Platinum-DNA Adducts Levels. About 107
cells were
collected and then lysed in 2 ml of 100 mM Tris⅐HCl, pH 8.5͞5
mM EDTA͞0.2% SDS͞200 mM NaCl͞100 ␮g/ml proteinase K.
The samples were gently agitated overnight at 55°C in the
incubator. The samples were extracted twice with 2 ml of
phenol͞chloroform͞isoamyl alcohol and followed by two extractions
with chloroform͞isoamyl alcohol. Genomic DNA was
precipitated with 2 ml of isopropanol, and the DNA was removed
and dissolved in 10 mM Tris⅐HCl (pH 7.5) buffer. The DNA was
treated with RNase and quantitated by UV-visible spectroscopy.
The amount of platinum on the DNA was determined by using
a Perkin–Elmer HGA-800 AAnalyst 300 atomic absorption
spectrometer.
Results
HMG1 Overexpression in MCF-7 and Evsa-T Cells. For MCF-7 cells, a
mammalian breast cancer cell line, a single 10Ϫ7
M dose of
estrogen increases HMG1 mRNA level by 1.5- to 2.5-fold (36).
The exact mechanism of this up-regulation is not known, although
HMG1 overexpression may be necessary to facilitate
transcriptional activation of hormone-responsive genes. We find
by immunofluorescence that the elevated level of HMG1 mRNA
transcript corresponds to higher protein levels (Fig. 1 A–C).
After MCF-7 cells were treated with 10Ϫ7
M estrogen for 0.5 h,
the fluorescence signal from labeled nuclear HMG1 was much
stronger than in the untreated cells (Fig. 1B). MCF-7 cells
contain both ER and PR and thus also respond to progesterone
treatment (10Ϫ6
M), up-regulating HMG1 within 0.5 h (Fig. 1C).
Evsa-T is a breast cancer cell line that has PR but not ER.
Estrogen treatment had no effect on HMG1 levels in Evsa-T cells
(data not shown). Progesterone treatment in Evsa-T cells raised
HMG1 protein levels as detected by immunofluorescence but
required a 4-h treatment (Fig. 1, compare E and F) in contrast
to MCF-7 cells. The kinetics of HMG1 overexpression has also
been confirmed by Western analysis. The amount of HMG1
peaked at 1.4-fold within 2 h of estrogen treatment in MCF-7
cells and leveled off by 24 h; the amount of HMG1 increased by
Fig. 1. HMG1 levels in hormone-treated MCF-7 and Evsa-T cells detected by immunofluorescence. (A) MCF-7 controls. (B) MCF-7 treated with 10Ϫ7 M estrogen
(0.5 h). (C) MCF-7 treated with 10Ϫ6 M progesterone (0.5 h). (D) Evsa-T controls. (E) Evsa-T treated with 10Ϫ6 M progesterone (0.5 h). (F) Evsa-T treated with 10Ϫ6
M progesterone (4 h). (Bar ϭ 50 ␮m.)
He et al. PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 ͉ 5769
BIOCHEMISTRY
about 2-fold after a 4-h progesterone treatment in Evsa-T cells
(data not shown).
Sensitization of Cells to Cisplatin by Estrogen or Progesterone. To
assess further the involvement of HMG domain proteins in
mediating cisplatin cytotoxicity, we investigated the effects of
elevated HMG1 levels on the sensitivity of cells to the drug. As
initially anticipated from the repair shielding hypothesis, we
found that elevated HMG1 expression levels were indeed paralleled
by increased sensitivity toward cisplatin. In MCF-7 cells,
estrogen or progesterone treatment increased cisplatin sensitivity
about 2-fold, according to the cisplatin concentrations where
only 50% of the cells are viable (Fig. 2A). A combination of
estrogen and progesterone sensitized the cells to cisplatin by a
factor of 4 (Fig. 2A); this additive effect suggests that they
independently up-regulate HMG1. In Evsa-T cells, progesterone
treatment induced 1.5-fold sensitization toward cisplatin,
whereas estrogen treatment had no effect because of the absence
of ER (Fig. 2B). In addition, the cisplatin sensitivity of HeLa
cells, an ER-negative cervical cancer cell line, did not change
after estrogen treatment (Fig. 2C).
The timing of hormone treatment plays an important role in
the degree of sensitization of cells to cisplatin. Cotreatment of
estrogen and cisplatin caused 2-fold sensitization in MCF-7 cells;
pretreatment with estrogen for 24 h did not cause any sensitization
(data not shown). In Evsa-T cells, the effect of sensitization
by progesterone was not observed with cotreatment (data
not shown) but was 2-fold with a 2-h pretreatment of progesterone
(Fig. 2B). Carboplatin is an analogue of cisplatin that can
form similar bifunctional DNA adducts, because the products of
aquation are the same for the two compounds (37). Estrogen had
no effect on the carboplatin sensitivity of cells treated simultaneously
with the two reagents, but a 24-h pretreatment of
carboplatin followed by a 4-h estrogen treatment increased the
sensitivity by 2-fold (Fig. 3).
Effects of Hormones on Cell Proliferation and Sensitivity to Other
Cytotoxic Agents. Estrogen induces general cell proliferation (38)
and regulates human mammary epithelial cell morphogenesis
(39, 40). We have considered the possibility that the sensitization
of cells toward cisplatin may be a consequence of hormoneFig.
2. Cell survival assays. (A) The effects of estrogen and progesterone
cotreatment on cisplatin sensitivity of MCF-7 cells. MCF-7 cells were cotreated
with 2 ϫ 10Ϫ7 M estrogen, 2 ϫ 10Ϫ7 M progesterone, or both hormones with
cisplatin for 4 h. (B) The effects of hormones on cisplatin sensitivity of Evsa-T
cells. Evsa-T cells were pretreated with 10Ϫ7 M estrogen or 10Ϫ6 M progesterone
for 2 h before a 4-h cisplatin treatment. (C) The effects of 10Ϫ7 M estrogen
on cisplatin sensitivity of HeLa cells.
Fig. 3. The effects of estrogen on the carboplatin sensitivity of MCF-7 cells.
MCF-7 cells were pretreated with carboplatin for 24 h before the addition of
10Ϫ7 M estrogen for 4 h.
5770 ͉ www.pnas.org He et al.
induced cell proliferation. Accordingly, we investigated whether
the effects of hormone treatment conditions used in the cell
survival assays, 4-h exposure to estrogen or progesterone, affected
cell growth. FACS analysis revealed no change in cellcycle
profile after hormone treatment (Table 1). Cell proliferation
rate, determined in cell counting assays, was similarly
unaffected by the transient hormone treatments (data not
shown).
trans-Diamminedichloroplatinum(II) (trans-DDP) is a clinically
inactive isomer of cisplatin that forms DNA adducts not
recognized by HMG1 (15). The trans-DDP sensitivity of MCF-7
cells was unaffected by estrogen treatment (data not shown).
Calicheamicin is another cytotoxic agent that causes doublestranded
DNA cleavage (41) in a manner that does not involve
HMG1. The sensitivity of MCF-7 cells toward calicheamicin was
also unaffected by estrogen treatment (data not shown). We
conclude that the hormone treatment did not sensitize the cells
to all cytotoxic agents but only to cisplatin or carboplatin via a
pathway that involves HMG1, presumably by repair shielding.
Platinum-DNA Adduct Levels. It is possible that DNA platination
levels could be elevated as a consequence of hormone treatment,
as suggested by reports that active promoter sites are preferentially
platinated by the drug (42, 43). We have investigated the
bound-platinum levels on genomic DNA in cells treated with
steroid hormones by platinum atomic absorption spectroscopy.
MCF-7 cells were treated with a range of cisplatin concentrations
(0–100 ␮M) for 4 h and then immediately harvested, and
genomic DNA was extracted to evaluate the initial platination
levels. The platinum signals were not detected with the atomic
absorption spectrometer in samples treated with 10 ␮M, 20 ␮M,
or 50 ␮M cisplatin. For samples treated with 100 ␮M of cisplatin,
the rb values, defined as the number of platinum atoms per
nucleotide, were 3.48 ϫ 10Ϫ4
for control cells and 3.33 ϫ 10Ϫ4
for estrogen-treated cells. The results indicate that the platinum
adduct levels on genomic DNA are comparable in control cells
and cells treated with estrogen.
Discussion
HMG1 already exists at a high level, between 10,000 and 100,000
copies per cell (24, 44), and an excess amount of the protein is toxic
(45), which may explain why only a moderate level of overexpression
of HMG1 was observed in the present study. Why then does
the moderate up-regulation observed in this study increase drug
sensitivity? One possibility is that endogenous HMG1 proteins are
already involved in complexes with chromatin and transcription
factors. HMG1 transiently expressed as a scaffold to facilitate ERor
PR-mediated transcription may be more readily available to bind
to cisplatin-DNA intrastrand crosslinks. The 2-fold difference in
cisplatin sensitivity is in good accord with the approximately 2-fold
increase in HMG1 protein levels.
The timing of the hormone treatments reflects the kinetics of
HMG1 up-regulation and cisplatin aquation chemistry. Cotreatment
of estrogen and cisplatin is more effective in sensitizing
MCF-7 cells than pretreatment for 24 h (data not shown). HMG1
protein levels increase within 0.5–2 h after estrogen treatment in
MCF-7 cells, whereas cisplatin may take a few hours to enter the
cell, undergo aquation, and bind to DNA (46). By the time
platinum-DNA damage occurs, HMG1 levels are high and can
block nucleotide excision repair efficiently. When cells are
pretreated with estrogen for 24 h, the amount of HMG1 has
leveled off at the time that platinum-DNA adducts form, and
consequently, the sensitization effect is less. On the other hand,
pretreatment of estrogen for 24 h sensitizes MCF-7 cells toward
carboplatin more than cotreatment. This observation is readily
explained by the difference in the rate of DNA binding for the
two drugs. To obtain the same degree of DNA damage, it is
necessary to use a 7.5-fold longer incubation time for carboplatin
than for cisplatin (47–49), owing to the slow rate of aquation of
the former. This difference may account for the required
preincubation period. In contrast to MCF-7 cells, HMG1 levels
increase in Evsa-T cells only after 4 h of progesterone treatment.
The effect of sensitization by progesterone is not observed with
cotreatment but is 2-fold greater with 2-h pretreatment of
progesterone.
Steroid hormones can induce higher transcriptional activities of
cell-cycle control genes (39, 40) and could trigger imbalance in the
cells and subsequent cisplatin sensitization. The transient steroid
hormone treatment does not change the cell growth and proliferation
characteristics of MCF-7 cells, according to FACScan analysis
and cell-counting assays. Progesterone does not cause cell proliferation
but still evokes the same sensitivity of cells to cisplatin. In
addition, the sensitivity toward other cytotoxic agents, such as
trans-DDP and calicheamicin, is not affected by hormone treatments.
The above evidence argues against higher transcriptional
activity being the main reason for the enhanced platinum-induced
toxicity of treated cells.
Another hypothesis that active promoter sites are preferentially
platinated by cisplatin was also investigated. Such sites
should be equally accessible to cisplatin and trans-DDP, however,
and the trans-DDP sensitivity was not influenced by
hormone treatment. In addition, platinum atomic absorption
data show similar levels of platinum-DNA adducts on genomic
DNA from estrogen-treated and control cells.
In summary, all available evidence supports the hypothesis
that hormone receptors are essential for HMG1 up-regulation
and subsequent cisplatin͞carboplatin sensitization in the cells.
Cisplatin produces many side effects including nephrotoxicity,
neurotoxicity, and emesis (50). Our study shows that the potency
of cisplatin can be increased through hormone treatment. From
a clinical perspective, estrogen and͞or progesterone treatment
should allow the currently applied cisplatin regimens to show
increased cytotoxicity toward cancer cells. Even a factor of 2
could be quite important in medical applications. In addition, the
enhanced potency of the drug may be sufficient to overcome
some acquired and intrinsic resistance. Many breast and ovarian
tumors express high levels of ER, PR, or both (51, 52), and these
cancers would be good candidates for cisplatin͞carboplatin
treatment in conjunction with hormone therapy. Because estrogen
has been implicated in the etiology of breast cancer owing
to its proliferative properties, progesterone may be preferred for
patients with breast cancer. For patients with ovarian and
cervical cancer, carboplatin is the standard chemotherapeutic
agent because of its diminished nephrotoxicity compared with
that of cisplatin (37). Even though pathways other than repair
shielding by HMG1 could be responsible for the observed
sensitization, this work establishes the potential for treating
patients with ovarian cancer with estrogen͞progesterone in
combination with carboplatin in clinical trials.
We thank J. M. Essigmann for access to tissue culture facilities and the
Massachusetts Institute of Technology Center for Cancer Research for
providing the FACS facilities. We greatly appreciate help received from
R. Hynes and J. Trevithick in conjunction with the immunofluorescence
Table 1. Cell-cycle analysis of MCF-7 cells treated with steroid
hormones
Cell treatment conditions
Percentage
G0–G1 S G2–M
N,N-dimethylformamide 4 h,
wash, wait 24 h
57 35 8
Estrogen 4 h, wash, wait 24 h 56 35 9
Progesterone 4 h, wash, wait 24 h 54 37 9
He et al. PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 ͉ 5771
BIOCHEMISTRY
study. The HeLa cell line was a gift from T. de Lange (Rockefeller
University, New York), and the Evsa-T cell line was from G. Leclercq
(Institut Jules Bordet, Brussels). This work was supported by a grant
from the National Cancer Institute (to S.J.L.), by the Howard Hughes
Medical Institute (to Q.H.), and by the Ludwig Cancer Research
Fellowship (to C.H.L.).
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