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Departments of Internal Medicine (W.-S.S, W.Y, J.-P.W., R.J.S.) and Health Evaluation Sciences (M.C.) University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; Department of General Surgery (S.M.), Keio University School of Medicine, Tokyo, Japan; and Laboratory of Cell Growth Regulation (R.K.), University of Texas MD Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Richard J. Santen, Division of Endocrinology, Department of Medicine, Box 334, University of Virginia Health Science Center, Charlottesville, Virginia 22908. E-mail: rjs5y{at}virginia.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise physiologic events responsible for sequential hormonal responses are not clearly understood. Clinical observations suggest that development of hypersensitivity to E2 may serve as one of the adaptive mechanisms. For example, premenopausal women initially experience tumor regression in response to surgical oophorectomy, an ablative therapy that decreases plasma E2 to levels of 10–20 pg/ml from basal of 50–600 pg/ml (4). Tumors later re-grow in response to these reduced levels but regress again when E2 is lowered further to 1–5 pg/ml with administration of aromatase inhibitors (5). The ability to re-grow at levels of 10–20 pg/ml when tumors initially required 50–600 pg/ml suggests the phenomenon of adaptive hypersensitivity.
Experimental observations in vitro provide support for the adaptive hypersensitivity hypothesis. A number of groups, in addition to our own, have studied the responses of cultured breast cancer cells to long-term E2 deprivation (6, 7, 8, 9, 10, 11, 12, 13, 14). In this and in other publications, we call these LTED (long-term deprivation of estradiol) cells. Results from several groups uniformly demonstrate an initial inhibition of growth when E2 is acutely removed from the culture media but resumption of cellular proliferation 1–3 months later. Under these conditions, LTED cells cannot be stimulated further with exogenous E2 but can be inhibited with pure antiestrogens (15).
Our previous data suggest that LTED cells have adapted to allow maximal growth in response to the small amounts of residual estrogen remaining in the culture media after a standard charcoal stripping procedure (15). When this residual E2 is completely removed from the media or antagonized by the pure antiestrogen, ICI 182780, LTED cells can then be stimulated to grow with exogenous E2. Furthermore, under these conditions, LTED cells respond to 2–4 log lower concentrations of E2 than wild-type, an observation that directly demonstrates hypersensitivity (16). This ability to shift dose response curves to the left by hormonal deprivation is not limited to breast cancer cells. LnCAP prostate cancer cells also grow in response to 1–2 log lower concentrations of androgen after deprivation of this sex steroid long term (17, 18). Taken together, these observations support the concept that cancer cells can adapt to their environment by developing mechanisms allowing growth in response to lowered levels of sex steroid.
A complementary study has examined the mechanisms involved in re-growth of MCF-7 cells after long-term E2 deprivation in vitro and demonstrated enhancement of MAP kinase activation (our unpublished observation). MAP kinase is an enzyme that catalyzes the phosphorylation of proteins involved in mediation of cell proliferation such as Elk-1 but which also mediates phosphorylation of the estrogen receptor (1, 19). Cross-talk between estrogen receptor-mediated and MAP kinase-stimulated effects on cell proliferation could potentially explain adaptive changes in level of sensitivity to E2 (1, 19). We demonstrated that MAP kinase plays a key mechanistic role in the regrowth phenomenon because blockade of MAP kinase activation markedly inhibits the proliferation of LTED cells grown in standard charcoal stripped medium (our unpublished observation).
The present study sought to demonstrate that hypersensitivity to E2 and activation of MAP kinase occurs under more complex in vivo conditions in LTED cells. Our experimental design enabled precise assessment of in vivo hypersensitivity. An E2 clamp method maintained plasma E2 at a series of doubling dose levels in oophorectomized nude mice (15). Measurement of wild-type and LTED xenograft volumes over a 2-month period served as the end point of tumor growth response. As evidence of hypersensitivity, we found that LTED xenograft tumors grew to a greater extent than wild-type in response to E2 concentrations of 1.25 and 2.5 pg/ml. Enhanced activation of MAP kinase characterized all LTED cell tumors regardless of estrogen dose. These in vivo data support the hypothesis that long-term E2 deprivation results in cells that are hypersensitive to E2 and contain a higher fraction of cells in which MAP kinase is activated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and inoculation into mice
Wild-type MCF-7 cells originally provided by Dr. Robert
Brueggemerier, Ohio State University were used for xenografts.
Conditions used to develop LTED cells as well as cell culture and
inoculation methods were as described previously (15, 20). Cultured
cells were removed from plates by scraping with a rubber policeman and
then resuspended in phenol-red free Matrigel (10 mg/ml; Becton Dickinson and Co., Bedford, MA). A total of 5 million cells (0.1
ml)/site was inoculated into each site. The MCF-7 cells were placed on
one side of the mouse at superior and inferior sites and LTED cells at
two identical sites on the contralateral side (Fig. 1
). Tumors were allowed to become established
for 1 month in the absence of exogenous E2 before
E2 implants were inserted. These experiments were
enabled by using the technique of Yue et al. (20), which
involved addition of Matrigel to tumor suspensions before implantation.
This technique markedly increases tumor "take" and allowed 100% of
LTED cell tumors to take (i.e. 90 out of 90) vs.
95% of wild-type tumors (i.e. 87 out of 90). At the end of
the 1-month period, tumor volumes did not differ significantly between
groups (i.e. 48.7 ± 16.7 mm3 for
the wild-type and 52.4 ± 15.2 mm3 for the
LTED tumors.) This approach allowed assessment of the effects of
E2 specifically on cell growth without the
confounding effects of estrogen on the initial establishment of
xenograft cells.
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Measurement of tumor growth
Growth rate was determined by measuring tumor length and width
with calipers every week for 2 months. Tumor volumes were estimated
according to the formula 4/3
r12r2
(r1; short axis, r2; long
axis). At the end of the 2-month period, animals were killed and
uterine weights measured. To allow for differences in tumor volumes at
the time of E2 implant insertion, results were
expressed as log differences from baseline in each of the four tumors
per animal.
Immunohistochemistry
An indirect immunoperoxidase method was performed to identify
activated MAP kinase. Tissues were fixed in 10% neutral buffered
formalin for 24 to 48 h and embedded in paraffin. Five-micrometer
sections in 10 mM citric buffer (pH 6.0) were heated for 20
min with a microwave (900 W, high power) for antigen retrieval, washed
in PBS, and treated with 0.3%
H2O2 in methanol to quench
the endogenous peroxidase activity. Sections were blocked with Avidin
D/Biotin blocking solutions and incubated with appropriate 10% normal
serum. Sections were then incubated with primary antibodies. Activated
(dually phosphorylated) MAP kinase was detected using mouse monoclonal
anti-activated MAP kinase IgG (1:500, Sigma, St. Louis,
MO) or rabbit polyclonal antiactivated MAP kinase IgG (1:600,
Quality Controlled Biochemicals, Inc., Hopkintown, MA).
These antibodies recognize only biphosphorylated (amino acid 202 and
204) active MAP kinase and not nonphosphorylated inactive enzyme (21, 22). Both of the antibodies recognizing MAP kinase have been validated
for specificity previously in studies using human tissues (21, 22).
Control sections were incubated without primary antibodies. Sections
were then incubated with appropriate secondary biotinylated antibodies
at 1:1000 dilutions followed by incubation with avidin-biotin complex
(ABC; Vector Laboratories, Inc., Burlingame, CA).
Immunoreactivity was visualized with DAB substrates. Sections were
counterstained with Harris hematoxylin.
In vitro experiments had demonstrated that activated MAP kinase increases in LTED cells compared with wild-type whereas total MAP kinase does not (our unpublished observations). To confirm this in vivo, we used an antibody that recognizes total MAP kinase (Zymed Laboratories, Inc., San Francisco, CA) and quantitated the percent of LTED and wild-type tumor cells that were total MAP kinase positive.
We attempted to provide additional evidence regarding activated MAP kinase by running Western blots of tumor homogenates. The results were confounded by the large amounts of activated MAP kinase present in the fibroblast capsules of tumors as demonstrated by immunohistochemistry (data not shown). Attempts at dissection of capsules away from tumors before preparing homogenates were not successful due to the fact that tumors were frozen immediately after harvesting.
Quantitation of immunohistochemical staining
Individual tumor cells were visually scored as positive or
negative based upon degree of staining under the light microscope. To
ensure randomization of fields to be counted, a series of squares were
outlined by a square reticule measuring 500 µ on each side that was
placed in the microscopic lens. Contiguous squares were counted
starting at a superior field and sequentially moving inferiorly until
the tumor capsule was reached. The square immediately to the right was
then counted and additional squares proceeding superiorly until a total
of approximately 1,000 cells was counted in each tumor. The percentage
of positive cells was then calculated.
Statistics
F tests based on the one-way ANOVA were used to
compare uterine weights among the groups. Volumes of the wild-type and
LTED tumors were transformed by calculating their logarithms. Paired
analyses were done by subtracting the log LTED tumor weight from the
log wild-type tumor weight for each animal and using one-way ANOVA on
the differences on the log tumor weights. As a check on the assumptions
for the ANOVA, Kruskal-Wallis nonparametric tests (23) were used to
compare uterine weight among the groups. Similar results were obtained
with the parametric and nonparametric tests.
Within each animal, two wild-type and two LTED tumors (Fig. 1
) were
measured weekly for 8 weeks, beginning at 1 month post inoculation. The
observed weekly tumor volumes were normalized by taking the logarithm
of the ratio of the weekly tumor volume divided by the corresponding
tumor volume at the first observation time. For the analyses of tumor
volume, two wild-type and two LTED normalized tumor volumes were
averaged for each animal. To compare the normalized tumor volume among
groups, the area under the normalized tumor volume curve for the
wild-type and LTED tumors were computed. Initial paired analyses were
done by subtracting the area under the wild-type tumor curve from the
area under the LTED tumor curve for each animal and using the
differences in a one-way ANOVA. Repeated measures models were also
applied to the weekly-normalized tumor volumes. These models allow for
comparing the groups with respect to the volumes of wild-type and LTED
tumors and account for the association between the two tumors growing
within an animal. The analyses reported in this paper are based on a
model that allows for the correlation between the tumor volume at two
observation times to be a decreasing function of the amount of time
between the two measurements. Other assumptions about the form of the
repeated measure model were also considered; similar analyses were
obtained under a variety of assumptions about the form of the
association between tumor volumes within animals. Logistic regression
models were used to compare the proportion of activated MAP Kinase
cells among the treatment groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These data demonstrated the linear
relationship between the ratio of E2/cholesterol
inserted into the SILASTIC brand implants and the plasma
E2 levels achieved.
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, we detected
an excellent linear dose-response relationship between the predicted
level of plasma E2 and stimulation of uterine
weight. To estimate the physiologic levels of E2
in intact animals, we measured uterine weight in
nonoophorectomized nude mice. These animals had a mean uterine
weight of 66.1 ± 9.1 mg, indicating that physiologic levels
approximate 1–3 pg/ml in intact animals. Taken together, these data
confirm the ability to clamp plasma E2 via
SILASTIC brand implants at levels bracketing physiologic concentrations
in vivo.
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. In the vehicle control groups
(i.e. oophorectomized animals with SILASTIC brand implants
containing cholesterol only), the LTED and wild-type MCF-7 cell
xenografts grew at rates that could not be distinguished statistically
(P = 0.38). At the 1.25 and 2.5 pg/ml doses, LTED
tumors grew to a greater extent than did wild-type (P =
0.003 and P = 0.0002, respectively). The lower
threshold response of LTED cells to E2 provides
direct evidence of hypersensitivity.
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. At the 5.0 pg/ml dose, both LTED and wild-type
xenografts increased their rate of growth to the same extent and
differences between these two groups were Aa–Ac).
Activated MAP kinase-containing cells were recognized as those with
strongly positive brown color staining. On inspection, the
LTED tumors appeared to contain more activated MAP kinase than did the
wild-type tumors. To provide further evidence of specificity, a
mono-specific polyclonal antibody was also used and demonstrated a
similar pattern of staining (Fig. 5, Ad–Af). Control tissues handled
identically but with omission of the primary antibody yielded minimal
background and no cellular staining (Fig. 5, Aa and Ad).
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). Further, the greater activation of MAP kinase
appeared to be an innate characteristic of the LTED tumors rather than
an effect of E2. Notably, no increase in MAP
kinase activation was apparent in response to E2
in the LTED tumors. In contrast, increasing doses of
E2 appeared to cause an increase in the
percentage of MAP kinase-positive cells in wild-type tumors.
To more precisely evaluate differences in number of MAP kinase positive
cells, we then carried out quantitative morphometry. We detected
activated MAP kinase with a range of 18.49% to 25.70% of cells in
LTED tumors and 2.66 to 6.40% of cells in wild-type tumors (Table 1). Notably, no overlap was seen between
LTED and wild-type tumors at each dose of E2
administered. The difference between the mean value for LTED and
wild-type groups was significant at the P < 0.0001
level. These data confirmed the clear enhancement of MAP kinase
activity in the LTED tumors.
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, P < 0.0001) in wild-type cells. In the vehicle control
animals, the average percent was 2.66 ± 0.39% and this increased
to a maximum of 6.40 ± 0.56% at the 20.0 pg/ml dose. Preliminary
data demonstrated no differences between wild-type and LTED tumor cells
with respect to the expression of total MAP kinase as previously
observed in our in vitro experiments (our unpublished
observations).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise mechanisms responsible for enhanced E2 sensitivity remain unclear but suggest the role of MAP kinase activation. Our in vitro studies as well as those of others (28) demonstrated an increase in MAP kinase activity in LTED cells. Cross-talk between estrogen and MAP kinase pathways acting at one of several potential levels might provide a mechanism responsible for E2 hypersensitivity (28, 29). Based upon these concepts, we considered it pertinent to determine whether MAP kinase activity is enhanced in LTED cells in vivo. Immunohistochemical analysis provided a precise means of addressing this issue. Both qualitative and quantitative assessment of the percent of cells containing activated MAP kinase clearly established an increase in LTED tumors. This occurred independently of E2 exposure in the LTED tumors because all tumors, regardless of exogenous E2 dose, had similar but increased levels of activated MAP kinase. Both in vitro (our unpublished observations) and in vivo results (data not shown) indicate that the increase is exclusively in the amount of activated but not total MAP kinase present. These data suggest that MAP kinase is activated by factors other than estrogens in LTED cells.
A variety of extracellular mitogens including growth factors, hormones, and cytokines can stimulate the activation of MAP kinase (30). Several investigators have demonstrated that long-term E2 deprivation of breast cancer cells results in an increase in secretion of growth factors or over-expression of growth factor receptors (8, 31, 32). After binding to their respective membrane receptors, growth factors initiate a cascade of effects. These include receptor phosphorylation, stimulation of the isoprenylation of RAS, increase in levels of RAF, and finally, activation of MAP kinase. Thus, an increase in growth factor receptor levels or in growth factor secretion could be responsible for activation of MAP kinase in the LTED cells. In vitro data in the complementary manuscript indicate that inhibitors of growth factor mediated tyrosine phosphorylation as well as isoprenylation of RAS block the proliferation of LTED cells (our unpublished observations). These observations support the concept that activation of growth factor pathways is involved in MAP kinase activation in LTED cells.
A key question is whether activated MAP kinase is mechanistically
involved in the enhancement of hypersensitivity to
E2 in LTED cells. Preliminary data from our group
suggest that this is the case. In a proof of principle experiment, we
have induced the activation of MAP kinase in wild-type MCF-7 cells by
exposing them to TGF
and examining sensitivity to
E2 under these conditions (16). This treatment
increases MAP kinase as detected on Western blot and causes a 2-log
enhancement of sensitivity to E2. This response
reflects an action of MAP kinase and not an unrelated effect of TGF.
This conclusion is based upon the observation that blockade of TGF
induced MAP kinase with a specific inhibitor, PD 98059, shifts the
dose-response curve back to baseline. We postulate from this series of
experiments that cells adapt to long-term E2
deprivation by increasing growth factor signaling pathway activation
and consequently the levels of activated MAP kinase.
The precise mechanism whereby MAP kinase may mediate enhanced sensitivity to E2 remains speculative. MAP kinase catalyzes the phosphorylation of the estrogen receptor and could act through the estrogen receptor to increase sensitivity to E2 (19, 29). Data (our unpublished observations) suggest that this is not the case because LTED cells are not hypersensitive to E2 with respect to ER transactivation (our unpublished observations). This conclusion is based upon the comparative assessment of dose dependent stimulation by E2 of ERE reporter activity and c-myc levels in wild-type and LTED cells. Based upon these observations, we believe that MAP kinase acts downstream of estrogen receptor mediated transcription to enhance estrogen mediated effects on cell proliferation.
Our observations suggest that growth factor pathways acting through MAP
kinase play a key role in the proliferation of LTED cells. The
hypersensitivity to E2, observed both in
vitro and in vivo, suggest an important role for
cross-talk between the ER-dependent and the growth factor pathways in
these cells. A potential mechanism for this interaction is the
ligand-independent activation of ER (19, 28, 29, 33, 34). This
process has been invoked to explain how peptide growth factors can
stimulate estrogen responsive tissues but still be subject to
inhibition by anti-estrogens. Ligand independent activation of steroid
hormone receptors by growth factors has been studied in various model
systems (33, 35). For example, it has been shown that the proliferative
activity of epidermal growth factor (EGF) is mediated via interaction
with the ER and that this effect can be antagonized by pure
anti-estrogens (33). This is further supported by the fact that EGF is
unable to stimulate uterine growth in ER-knockout mice (36). These
data, taken together, suggest that the ER and growth factor pathways
can interact. Data presented in the complementary manuscript, suggest
that MAP kinase is involved in E2 stimulated
proliferation but may not be important in the absence of exogenous
E2 (our unpublished observations).
To add to the complexity, estrogens can stimulate growth factor production and consequently MAP kinase activation (37, 38). This is the likely explanation for the stimulation of MAP kinase with E2 in wild-type xenografts. Taken in the context of our studies, the cross-talk between receptor mediated and growth factor pathways could potentially diminish the concentration of E2 required to stimulate cell proliferation and explain the hypersensitivity to E2 observed in LTED cells in vitro and in vivo. Clearly further studies are required to sort out these highly complex interactions.
As we had shown in vitro (15), the present study
demonstrates that LTED xenografts grow to a greater extent than
wild-type tumors in response to very low concentrations of
E2. In contrast, higher E2
concentrations cause greater growth of wild-type than LTED tumors. From
clinical observations, it has been known that E2
can exert both stimulatory and inhibitory effects on breast tumor
growth. Low doses of E2 stimulate tumor growth,
whereas high doses of diethylstilbestrol cause tumor regression (1).
Cross-over responses to DES in patients relapsing after tamoxifen as
well as responses to tamoxifen in DES failures have been observed (39).
The mechanisms for these inhibitory effects of E2
have not been explained and could involve participation of
co-activators or co-repressors of receptor transcription or other
mechanisms. This phenomenon can be mimicked in vitro with
increasing concentrations of E2, which elicit a
bell shaped dose-response curve. Cell growth is initially stimulated by
increasing doses of E2 until peak growth is
achieved. Further increases in E2 concentration
then begin to diminish growth (15). In vitro, the LTED cells
appear to be hypersensitive to both the stimulatory and inhibitory
components of E2. In our in vivo
observations, very low doses of E2
(i.e. 1.25 and 2.5 pg/ml) initially stimulate the LTED cells
to a greater extent than wild-type (Fig. 4A). At the transition dose of
5.0 pg/ml, no differences are observed between LTED and wild-type tumor
growth. At the higher 10.0 and 20.0 pg/ml doses, the wild-type cells
are stimulated to a greater extent than LTED (Fig. 4B). This
observation may reflect the initial appearance of some inhibitory
effects of E2 in the LTED cells. Further data
will be required to determine if this is the case and to evaluate the
mechanisms involved.
The growth curves in our xenograft model, while highly reproducible, demonstrate a pattern of initial growth followed by a plateau or partial regression. The explanation for this pattern is not understood. Re-implantation of SILASTIC brand implants at week 4 should allow maintenance of E2 at expected concentrations for the full 8-week period. Prior data in our laboratory demonstrated that the SILASTIC brand implants maintain E2 for at least 4 weeks (unpublished data). Tumor growth represents an increase in the rate of cell proliferation over that of apoptotic cell death. It will be necessary now to determine whether these xenografts begin to undergo an increased rate of apoptosis between weeks 4 and 8.
With other MCF 7 xenografts, we have observed continued growth of tumors to volumes substantially higher than in these experiments (24). Thus, we do not believe that the plateau represents growth beyond the ability to increase vascular supply. While additional experiments are required to fully understand this phenomenon, its presence does not confound interpretation of our data on effects of E2. The use of the integrated tumor volume method allows us to precisely determine the effects of E2 even in the presence of the plateau effect.
Our methodology allowed establishment of tumors and minimal growth in the absence of exogenous E2 in castrated nude mice. Other investigators (8) have implanted wild-type MCF-7 cells into castrated nude mice without exogenous estrogen supplementation and found only transient tumor establishment followed within 20 days by complete regression. Our results differ in that we achieved 96.7% of wild-type tumor establishment and no regression thereafter in absence of E2. These differing results probably reflect the ability of the Matrigel implantation method to establish viable tumors effectively without exogenous estrogen. Without Matrigel, less than 10% of wild-type tumors were established and grew in the absence of E2 (15).
In about one-third of breast cancers, estrogen stimulates tumor growth, whereas estrogen deprivation causes tumor regression. Inhibition of estrogenic stimulation is the main target for endocrine treatment of breast cancer (1). Primary treatment with oophorectomy or tamoxifen in premenopausal women and tamoxifen in postmenopausal frequently causes tumor regressions. Responses to secondary hormonal therapies such as aromatase inhibitors, surgical adrenalectomy or hypophysectomy, or synthetic progestins have been well documented but never explained mechanistically. Data presented in this study provide evidence for at least one mechanism to explain secondary responses, the development of hypersensitivity to E2. This mechanism would explain why responsiveness to agents which effectively inhibit E2 production such as the aromatase inhibitors, are active as second line therapy in previously oophorectomized patients. Secondary responses to aromatase inhibitors following relapse on tamoxifen could be similarly explained but will require evidence that tamoxifen also induces E2 hypersensitivity.
Because of relative assay insensitivity, prior studies have been unable to precisely define the levels of E2 in intact nude mice (24). These animals lack testosterone-estrogen binding globulin (TEBG), and thus nearly all of the E2 circulates as free hormone (40). In other species that lack TEBG, such as the ewe, plasma E2 averages 2–3 pg/ml (40). In the ewe, it is not difficult to obtain large amounts of plasma to extract for estrogen RIA and precise measurements of plasma estrogen levels have been made. This is not possible in the nude mouse and definitive measurements of basal E2 levels are lacking. Of interest is the fact that a uterine bioassay provides a more sensitive means of assessing basal E2 levels in intact mice than RIA. With this technique, we were able to estimate basal levels to be in the range of 1.25 to 2.5 pg/ml. These levels are capable of stimulating the growth of LTED but not wild-type xenografts.
Several issues regarding hypersensitivity remain unresolved and require further study in this in vivo model. One is the precise regulation of c-myc expression and whether or not the message and protein levels of c-myc are increased basally and in response to E2 in LTED vs. wild-type cells. Preliminary data from Western blot analysis suggest an increase of c-myc in LTED vs. wild-type cells under the basal conditions and an increase following E2 (data not shown). Further confirmation is required for this issue. Secondly, the effects of aromatase inhibitors and antiestrogens on growth of LTED tumors must be evaluated. Residual estrogen concentrations in LTED tumors could contribute to the basal elevations of activated MAP kinase. Finally, a clear mechanistic link between activated MAP kinase and hypersensitivity must be demonstrated in vivo.
In conclusion, this study directly demonstrates that long-term E2 deprived cells are hypersensitive to E2 when implanted as xenografts. Further, these cells exhibit increases in MAP kinase activity. These observations suggest a dynamic interplay whereby activation of MAP kinase renders cells more sensitive to the proliferative effects of E2. The precise mechanisms for this interplay are unknown but, when further understood, could potentially provide insight into approaches to prevent the evolution of tumors to a hormone-insensitive state.
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Acknowledgments |
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Footnotes |
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Received June 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and –ß production and cell
responsiveness during the progression of MCF-7 human breast cancer
cells to estrogen-autonomous growth. Cancer Res 54:5867–5874-induced but not estrogen-induced growth of
hormone-independent human breast cancer. Mol Endocrinol 2:543–555This article has been cited by other articles:
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L-A Martin, S Pancholi, C M W Chan, I Farmer, C Kimberley, M Dowsett, and S R D Johnston The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling Endocr. Relat. Cancer, December 1, 2005; 12(4): 1017 - 1036. [Abstract] [Full Text] [PDF]
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R J Santen, R X Song, Z Zhang, R Kumar, M-H Jeng, A Masamura, J Lawrence Jr, L Berstein, and W Yue Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S61 - S73. [Abstract] [Full Text] [PDF]
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C. M Staka, R. I Nicholson, and J. M W Gee Acquired resistance to oestrogen deprivation: role for growth factor signalling kinases/oestrogen receptor cross-talk revealed in new MCF-7X model Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S85 - S97. [Abstract] [Full Text] [PDF]
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R I Nicholson, C Staka, F Boyns, I R Hutcheson, and J M W Gee Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy Endocr. Relat. Cancer, December 1, 2004; 11(4): 623 - 641. [Abstract] [Full Text] [PDF]
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A. Ring and M. Dowsett Mechanisms of tamoxifen resistance Endocr. Relat. Cancer, December 1, 2004; 11(4): 643 - 658. [Abstract] [Full Text] [PDF]
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V. T. Gaddy, J. T. Barrett, J. N. Delk, A. M. Kallab, A. G. Porter, and P. V. Schoenlein Mifepristone Induces Growth Arrest, Caspase Activation, and Apoptosis of Estrogen Receptor-Expressing, Antiestrogen-Resistant Breast Cancer Cells Clin. Cancer Res., August 1, 2004; 10(15): 5215 - 5225. [Abstract] [Full Text] [PDF]
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L. M. Berstein, J.-P. Wang, H. Zheng, W. Yue, M. Conaway, and R. J. Santen Long-Term Exposure to Tamoxifen Induces Hypersensitivity to Estradiol Clin. Cancer Res., February 15, 2004; 10(4): 1530 - 1534. [Abstract] [Full Text] [PDF]
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 S. Toth-Fejel, J. Cheek, K. Calhoun, P. Muller, and R. F. Pommier Estrogen and Androgen Receptors as Comediators of Breast Cancer Cell Proliferation: Providing a New Therapeutic Tool Arch Surg, January 1, 2004; 139(1): 50 - 54. [Abstract] [Full Text] [PDF]
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R. J. Santen, R. X. Song, Z. Zhang, W. Yue, and R. Kumar Adaptive Hypersensitivity to Estrogen: Mechanism for Sequential Responses to Hormonal Therapy in Breast Cancer Clin. Cancer Res., January 1, 2004; 10(1): 337S - 345S. [Abstract] [Full Text] [PDF]
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 L.-A. Martin, I. Farmer, S. R. D. Johnston, S. Ali, C. Marshall, and M. Dowsett Enhanced Estrogen Receptor (ER) {alpha}, ERBB2, and MAPK Signal Transduction Pathways Operate during the Adaptation of MCF-7 Cells to Long Term Estrogen Deprivation J. Biol. Chem., August 15, 2003; 278(33): 30458 - 30468. [Abstract] [Full Text] [PDF]
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 J. M. Knowlden, I. R. Hutcheson, H. E. Jones, T. Madden, J. M. W. Gee, M. E. Harper, D. Barrow, A. E. Wakeling, and R. I. Nicholson Elevated Levels of Epidermal Growth Factor Receptor/c-erbB2 Heterodimers Mediate an Autocrine Growth Regulatory Pathway in Tamoxifen-Resistant MCF-7 Cells Endocrinology, March 1, 2003; 144(3): 1032 - 1044. [Abstract] [Full Text] [PDF]
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H. Kurokawa and C. L. Arteaga ErbB (HER) Receptors Can Abrogate Antiestrogen Action in Human Breast Cancer by Multiple Signaling Mechanisms Clin. Cancer Res., January 1, 2003; 9(1): 511S - 515S. [Abstract] [Full Text] [PDF]
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W. Yue, J.-P. Wang, M. Conaway, S. Masamura, Y. Li, and R. J. Santen Activation of the MAPK Pathway Enhances Sensitivity of MCF-7 Breast Cancer Cells to the Mitogenic Effect of Estradiol Endocrinology, September 1, 2002; 143(9): 3221 - 3229. [Abstract] [Full Text] [PDF]
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 A. M. Soto and C. Sonnenschein The Two Faces of Janus: Sex Steroids as Mediators of Both Cell Proliferation and Cell Death J Natl Cancer Inst, November 21, 2001; 93(22): 1673 - 1675. [Full Text] [PDF]
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R. X.-D. Song, G. Mor, F. Naftolin, R. A. McPherson, J. Song, Z. Zhang, W. Yue, J. Wang, and R. J. Santen Effect of Long-Term Estrogen Deprivation on Apoptotic Responses of Breast Cancer Cells to 17{beta}-Estradiol J Natl Cancer Inst, November 21, 2001; 93(22): 1714 - 1723. [Abstract] [Full Text] [PDF]
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 M. Maggiolini, D. Bonofiglio, S. Marsico, M. L. Panno, B. Cenni, D. Picard, and S. Ando Estrogen Receptor alpha Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Two Major Phytoestrogens on Human Breast Cancer Cells Mol. Pharmacol., September 1, 2001; 60(3): 595 - 602. [Abstract] [Full Text] [PDF]
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R. A. McClelland, D. Barrow, T.-A. Madden, C. M. Dutkowski, J. Pamment, J. M. Knowlden, J. M. W. Gee, and R. I. Nicholson Enhanced Epidermal Growth Factor Receptor Signaling in MCF7 Breast Cancer Cells after Long-Term Culture in the Presence of the Pure Antiestrogen ICI 182,780 (Faslodex) Endocrinology, July 1, 2001; 142(7): 2776 - 2788. [Abstract] [Full Text] [PDF]
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 R. Clarke, F. Leonessa, J. N. Welch, and T. C. Skaar Cellular and Molecular Pharmacology of Antiestrogen Action and Resistance Pharmacol. Rev., March 1, 2001; 53(1): 25 - 72. [Abstract] [Full Text] [PDF]
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I. Kuter Breast Cancer Update Oncologist, August 1, 2000; 5(4): 285 - 292. [Abstract] [Full Text]
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