From the Department of Pharmacology, Dartmouth
Medical School, Hanover, New Hampshire 03755, the ¶ Department of
Biochemistry and Molecular Biology, Medical College of Ohio, Toledo,
Ohio 43699-0008, and the
Department of Surgery, University of
Texas Health Science Center at San Antonio,
San Antonio, Texas 78284-7840
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ABSTRACT |
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In view of the tumor suppressor role of the
transforming growth factor- (TGF
) type II receptor (RII), the
identification and characterization of agents that can induce the
expression of this receptor are of potential importance to the
development of chemoprevention approaches as well as treatment of
cancer. To date, the identification of exogenous agents that control
RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGF
growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In
contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which
have lost cell surface RII and are resistant to TGF
, was not
affected by these two compounds. TGF
-neutralizing antibody was able
to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGF
activity. An RNase protection assay showed approximately a 3-fold induction of
the RII mRNA, while a receptor cross-linking assay revealed a
3-4-fold induction of the RII protein. In contrast, there was no
change in either RII mRNA or protein in the MCF-7 L cells.
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INTRODUCTION |
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Transforming growth factor-
(TGF
)1 comprises a family
of hormone-like polypeptides that affects cell growth, adhesion, and differentiation (1). They act as growth inhibitors for most epithelial
cells and some cancer cells. Two pathways are primarily involved in
mediating effects of TGF
on cell growth and differentiation. One
pathway involves blockade of cell cycle transit, while the other
involves alteration of the extracellular matrix environment.
TGFs elicit their effects by binding to cell surface receptors.
Three major types of receptors have been shown to be present in most
TGF
-responsive cell lines. They are designated as type I (RI), type
II (RII), and type III (RIII), respectively. RIII is a 280-330-kDa
glycoprotein that has no functional signaling domain but rather serves
as a ligand storage protein and presents TGF
to the signaling
receptors (2). RI and RII, which are glycoproteins of ~55 and 85 kDa,
respectively, form a heteromeric receptor complex. Both are
serine/threonine kinases, and each appears to be indispensable for
TGF
signaling (3-5). The direct involvement of both RI and RII in
conferring TGF
effects indicates that loss of either of the
functional receptors would contribute to loss of autocrine TGF
activity. Loss of negative autocrine TGF
activity results in a
growth advantage caused by an imbalance in positive and negative
regulators, possibly leading to tumor formation and progression (6, 7).
Recent evidence has shown a loss of RII is often associated with the
failure to respond to autocrine and exogenous TGF
. We have
previously demonstrated that re-expression of this receptor in an
RII-deficient breast cancer cell line (late passage MCF-7) leads to
restoration of TGF
sensitivity and reduced malignancy in athymic
nude mice (6). In addition, it has been shown that mutational
inactivation of RII occurs frequently in a subset of colon tumors with
microsatellite instability (7), and reconstitution of RII expression by
stable transfection also leads to reversal of malignancy in these cells (8). Others have noted that loss of RII expression is important in
other types of malignancies (9-15). These lines of evidence suggest
that RII is a critical determinant for conferring TGF
tumor
suppression as well as negative autocrine TGF
growth function. Consequently, agents that can induce RII expression would be valuable in the development of approaches for cancer treatment and prevention where receptor expression appears to be repressed, such as estrogen receptor-positive (ER+) breast cancer (16, 17). To date, no
such agents have been carefully characterized for their ability
to induce RII. Although Cohen et al. (55) showed increased
RII mRNA in a human neuroblastoma cell line after retinoic acid
(RA) treatment, they were not able to detect cell surface RII. In
addition, they failed to test for increased autocrine activity and
increased responsiveness/growth inhibition to TGF
after RA
treatment. A study by Turley et al. (56) in RL human B
lymphoma cells demonstrated increased RII protein levels
following treatment with RA and vitamin E succinate. However, they did
not investigate whether this correlated with increased levels of cell
surface RII.
Development of effective therapeutic and preventive approaches for breast cancer remains an issue, since conventional treatment by antiestrogens such as tamoxifen often leads to resistance in estrogen receptor-positive tumors (18), and chemotherapy of estrogen receptor-negative tumors is even less effective (19). Since there is a high incidence of vitamin D receptors (VDRs) in human breast cancer tumors (21, 22), vitamin D3 is an appealing candidate as a new therapeutic agent. Like other steroid hormones, it mediates its effect through interaction of its nuclear receptor (VDR) with DNA-responsive elements in the target genes (20). Moreover, many breast cancer cell lines are responsive to vitamin D3 antiproliferative effects both in vitro and in athymic mice (23). However, a major drawback for its clinical application is that the doses effective for suppressing tumor growth often cause hypercalcemia. Consequently, analogues have been developed to reduce the calcemic effects while increasing the potency of inhibition of proliferation (23, 24). Two analogues, EB1089 and MC903, both of which are derived by modification of the C17 side chain of vitamin D3, have been shown to be effective against rat breast tumors in vivo (24) or as an antiproliferative agent when given topically for psoriasis as well as for cutaneous metastatic breast cancer (25). However, the mechanisms of vitamin D3-mediated growth inhibition and in particular its anti-tumor action remain largely unresolved.
In this report, we show a correlation between RII expression and
vitamin D3 inhibition in MCF-7 sublines that differ
dramatically in their RII expression and hence their TGF sensitivity
as well. We hypothesized that vitamin D3's mechanism of
inhibition might involve induction of TGF
autocrine activity through
increased expression of RII. This hypothesis was confirmed by RNase
protection assays showing approximately 3-fold induction of the RII
mRNA and a 3-4-fold induction of cell surface RII protein. The
increased inhibition by vitamin D3/analogues was blocked by
TGF
-neutralizing antibodies, indicating an induction of negative
autocrine TGF
activity.
The use of an essential dietary nutrient with antiproliferative and
anti-tumor properties represents an attractive approach for
chemoprevention and/or therapy. This is particularly true of vitamin D
compounds, since the high stress western style diet associated with
colon and breast cancer is also associated with low levels of vitamin D
and calcium (26). Thus, increased autocrine negative TGF activity
mediated by vitamin D3 compounds in MCF-7 E cells may
provide a novel mechanism for blocking malignant progression by
chemopreventive approaches.
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MATERIALS AND METHODS |
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Cell Cultures-- MCF-7 E cells (passage number 150) were kindly provided by Drs. Robert J. Pauley and Herbert D. Soule from the Michigan Cancer Foundation. MCF-7 L cells were obtained from the ATCC and used at a passage number greater than 500. These cell lines were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, pyruvate, vitamins, amino acids, and antibiotics. Working cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2.
Vitamin D3 Compounds--
1,25-(OH)2
vitamin D3 as well as its analogues EB1089 and MC903 were
generous gifts from Dr. Lise Binderup of LEO Pharmaceutical Products
(Ballerup, Denmark). Stock solutions were prepared in isopropyl alcohol
at 4 mM. Serial dilutions were made in absolute ethanol and
stored at 20 °C protected from light. These diluted solutions were
added to the experimental culture media at a final ethanol
concentration of 0.1%. Control cells received 0.1% ethanol vehicle,
which had no effect on cell proliferation.
DNA Synthesis Assay--
[3H]thymidine
incorporation into DNA was measured as described previously to
determine TGF and vitamin D3 sensitivity (6). Briefly,
MCF-7 cells were seeded in 24-well tissue culture plates at a density
of 1.5 × 104 cells/well in 1 ml of medium. Various
concentrations of compounds (1,25-(OH)2 D3,
EB1089, MC903, or TGF
) were added after cell attachment
(approximately 2 h). Following 4 days of incubation, cells
received a 2-h pulse with [3H]thymidine (7 µCi, 46 Ci/mmol, Amersham Pharmacia Biotech). DNA was then precipitated with
10% ice-cold trichloroacetic acid, and the amount of
[3H]thymidine incorporated was analyzed by liquid
scintillation counting in a Beckman LS 7500 scintillation counter as
described previously (6). To determine whether there is an increase in the inhibitory effects by TGF
1 following EB1089
treatment, MCF-7 E cells, which are TGF
-responsive, were plated as
described above. Various concentrations of EB1089 plus 0.1 ng/ml of
TGF
1 were added after attachment. Cells were incubated
and [3H]thymidine incorporation was determined as
described above.
TGF-neutralizing Antibody Assay--
Cells were resuspended
at a concentration of 1.5 × 104 cells/ml and plated
into 24-well tissue culture plates (1 ml/well) either untreated or in
the presence of 10 µg/ml TGF
1 neutralizing antibody (R
& D Systems) or control normal IgG. After 3 h of incubation, different concentrations of vitamin D3 compounds were added
as indicated. Cells were allowed to grow for 72 h without changing the media, followed by determination of [3H]thymidine
incorporation as described above.
RNA Analysis--
RNase protection assays were performed to
determine RII RNA expression levels after vitamin D3
treatment. A 476-base pair fragment of the RII cDNA within the
cytoplasmic region was obtained by polymerase chain reaction with the
following primers: 5'-TGGACCCTACTCTGTCTGTG-3' and
5'-TGTTTAGGGAGCCGTCTTCA-3'. The fragment was subcloned into a pBSK ()
plasmid (Stratagene, La Jolla) for making the RII riboprobes. In
vitro transcription using T3 RNA polymerase yields antisense riboprobes that protect a 476-base pair RII fragment. RNase protection assays were performed as described previously (27). Briefly, exponentially growing cells were treated with EB1089 at 1 × 10
8 M for the indicated time periods. Cells
were solubilized in guanidine thiocyanate, and total RNA was obtained
by cesium chloride gradient ultracentrifugation (28). 40 µg of total
RNA was used for overnight hybridization with 32P-labeled
antisense riboprobes. Following RNase A and T1 treatment, the protected double-stranded RNA fragments were heat-denatured at
95 °C and analyzed by urea-polyacrylamide gel electrophoresis, and
the radioactive probes were visualized by autoradiography. Actin was
used as an internal control for normalizing the amount of sample
loading.
Receptor Cross-linking--
Simian recombinant
TGF1 was purified as described (29) and iodinated by the
chloramine T method (30). MCF-7 cells were seeded into
35-mm2 tissue culture wells at a density of 105
cells/well. In the kinetic studies, exponentially growing cells were
treated with various concentrations of the compounds for 24 h or
with a single concentration for the indicated time periods. Cell
monolayers were then incubated with 200 pM
125I-labeled TGF
1 at 4 °C for 4 h
followed by chemical cross-linking with disuccinimidyl suberate for 15 min (31). Labeled cell monolayers were solubilized in 200 µl of 1%
Triton X-100 with 1 mM phenylmethylsulfonyl fluoride. Equal
amounts of cell lysate protein were separated by 4-10% gradient
SDS-polyacrylamide gel electrophoresis under reducing conditions and
exposed for autoradiography.
Mink Lung Epithelial Cell Growth Inhibition Assay--
MCF-7 E
cells were plated into 100-mm2 tissue culture dishes and
allowed to reach 70-80% confluency. The medium was then removed and
replaced with 5 ml of fresh McCoy's 5A medium supplemented with
pyruvate, vitamins, amino acids, and antibiotics (SM). The cells were
then treated with EB1089 (1 × 108 M) or
with vehicle only for 24 h. Following treatment, the conditioned medium was collected, and the indicated volumes were used to treat mink
lung epithelial cells plated in 96-well tissue culture plates at a
density of 1500 cells/well. In addition, a standard TGF
growth
inhibition curve was generated by treating the cells with various
concentrations of TGF
1. The mink lung epithelial cells were allowed to incubate for 3 days at which time the medium was removed and replaced by 100 µl of fresh SM. Colonies were immediately visualized by staining with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma)
for 2 h. The stained cells were solubilized with Me2SO
(dimethyl sulfoxide) (Mallinckrodt) and the relative cell numbers were
then determined by the resultant absorbance at 595 nm.
Luciferase Assay--
The TGF-responsive cyclin A promoter in
tandem with a luciferase reporter construct (
133/
2) was used as
described previously (32). The reporter construct (
133/
2) contains
only the activating transcription factor site, which has been shown to
be the site required to mediate down-regulation of cyclin A promoter
activity by TGF
1 in mink lung epithelial cells (32).
MCF-7 E cells were transiently transfected with 30 µg of luciferase
reporter construct and 7 µg of
-galactosidase plasmid by
electroporation with a Bio-Rad gene pulser at 250 mV and 960 microfarads. Cells were plated into a six-well tissue culture plate and
treated with TGF
-neutralizing antibody (10 µg/ml) or control
normal IgG and allowed to attach for 3 h. Following attachment,
cells were treated with EB1089 (1 × 10
8
M), while control cells were treated with vehicle only. At
51 h post-transfection, cells were harvested with 100 µl of
lysis buffer (Luciferase assay system, Promega). Luciferase activity was determined according to the manufacturer's instruction using a
luminometer (Berthold Lumat LB 0501) and expressed as relative units
after normalization to
-galactosidase.
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RESULTS |
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TGF Sensitivity of MCF-7 Cells--
Inconsistent response of
MCF-7 cells to TGF
has been observed in several laboratories (34,
35), probably due to growth selection during long term passage of
cultures. Having obtained both early (150) and late (>500) passage
MCF-7 cells, we decided to first test whether they responded
differently to TGF
(Fig. 1). MCF-7 E
cells showed a significant dose-dependent inhibition by
TGF
with an IC50 of 0.2 ng/ml. In contrast, MCF-7 L
cells demonstrated complete resistance to TGF
up to 25 ng/ml (Fig. 1). As described below, MCF-7 E cells expressed RII mRNA and
protein in contrast to MCF-7 L cells, which had 5-fold less mRNA
and no detectable cell surface protein.
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Vitamin D3 Sensitivity--
Effects of
1,25-(OH)2 D3 and its analogues on cell
proliferation of MCF-7 cells were investigated by assessing
[3H]thymidine incorporation following treatment by these
compounds as described under "Materials and Methods." MCF-7 E cells
showed a dose-dependent inhibition by vitamin
D3 with an IC50 of 5 × 108
M. In contrast, MCF-7 L cells were not affected by vitamin
D3 (Fig. 2A).
Vitamin D3 analogues EB1089 and MC903 demonstrate similar growth-inhibitory
patterns (Fig. 2, B and C). The overall potency of growth inhibition by EB1089 was approximately 2 orders of magnitude higher than vitamin D3. MCF-7 E cells showed an
IC50 of 2.5 × 10
10 M, and
MCF-7 L cells did not respond to EB1089 treatment up to 1 × 10
7 M.
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TGF Autocrine Activity--
The correlation between TGF
and
vitamin D3 sensitivity suggested that vitamin
D3 may function through increasing TGF
autocrine-negative activity in MCF-7 E cells. To test this hypothesis,
TGF
-neutralizing antibodies were used to determine whether they were
capable of blocking the growth inhibition induced by these compounds
(Fig. 3). At 10 µg/ml,
TGF
1-neutralizing antibody reversed the inhibitory effect of vitamin D3 and its analogues, generating an
approximately 60% increase in DNA synthesis as compared with the
normal chicken IgG treatment. In contrast, MCF-7 L cells did not
respond to TGF
1-neutralizing antibody, indicating a lack
of induction of autocrine TGF
activity. These results indicate that
the growth-inhibitory mechanism of vitamin D3 involves
induction of TGF
autocrine-negative activity in MCF-7 E cells.
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Alteration of RII Expression--
Increased autocrine TGF
activity could result from enhanced expression of TGF
isoforms
and/or their receptors. To test these possibilities, RNase protection
assays were initially carried out on MCF-7 E cells to determine whether
there were alterations of TGF
isoform expression upon treatment with
vitamin D3 compounds. MCF-7 E cells expressed high levels
of TGF
1 mRNA and low levels of TGF
2
and TGF
3 mRNA. Treatment with EB1089 did not
generate altered mRNA expression for any of the three TGF
isoforms (data not shown). In addition, enzyme-linked immunosorbent
assay analysis of the conditioned medium showed no significant increase
in the levels of activated TGF
1 protein (data not
shown). Since the levels of activated TGF
cannot be determined by
enzyme-linked immunosorbent assay analysis, a growth inhibition
bioassay on mink lung epithelial cells was performed. The condition
medium from EB1089-treated and -untreated MCF-7 E cells was added to mink lung epithelial cells as described under "Materials and
Methods." After exposure to either treated or untreated conditioned
medium, no significant difference in growth inhibition was observed in the mink lung epithelial cells (Fig. 4).
These results indicate that EB1089 treatment did not alter the
activation of secreted growth and/or inhibitory peptides from MCF-7 E
cells, one of which is likely to be TGF
1, as
demonstrated by enzyme-linked immunosorbent assay analysis. Taken
together, these results suggest that the enhanced TGF
autocrine
activity by vitamin D3 did not result from modulation of
ligand expression or activation.
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Responsiveness to Autocrine TGF--
To evaluate whether RII
induction by treatment with EB1089 enhanced autocrine TGF
sensitivity, TGF
-dependent promoter activity was
analyzed using the TGF
-responsive cyclin A luciferase reporter construct (32). TGF
induces down-regulation of cyclin A promoter activity but requires a functional TGF
type I and II receptor complex (32, 33). Thus, an increase in functional receptor levels would
result in enhanced down-regulation of cyclin A promoter activity. The
cyclin A reporter construct (
133/
2) contains only the activating
transcription factor site, which has been shown to mediate
down-regulation of cyclin A promoter activity by TGF
1 in
mink lung epithelial cells (32). This reporter construct was
transiently transfected into MCF-7 E cells, which are sensitive to
TGF
, followed by treatment with TGF
-neutralizing antibody and
EB1089 as described under "Materials and Methods." As expected, a
decrease in luciferase activity was induced in MCF-7 E cells following
treatment with EB1089. TGF
-neutralizing antibody reversed the
decrease in cyclin A luciferase activity by EB1089, increasing it by
approximately 70%. TGF
-neutralizing antibody alone had no
significant affect on cyclin A luciferase activity (Fig.
8) The fact that EB1089 did not increase
expression or activation of any of the three TGF
isoforms indicates
that enhanced responsiveness to autocrine TGF
after EB1089 treatment
is due to the increased expression of RII.
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DISCUSSION |
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TGF signaling requires a heteromeric assembly of its two
Ser-Thr kinase receptors, designated RI and RII, respectively (36). A
recent model illustrating the physical and functional interactions between the two receptors proposes that upon ligand binding, the constitutively active RII recruits RI and transphosphorylates the RI,
which subsequently initiates downstream cytoplasmic events (37).
Defects in expression of either receptor would contribute to loss of
response to exogenous as well as endogenous TGF
. In particular, loss
and/or lack of autocrine TGF
response plays a major role in
enhancing tumor progression. As was demonstrated previously, antisense
TGF
transfection in two early malignant colon carcinoma cell lines
eliminated autocrine negative TGF
activity but did not block
response to exogenous TGF
(27, 38). The TGF
antisense-transfected
cells showed increased tumor growth and incidence in athymic nude mice,
indicating that autocrine TGF
plays a key role in blocking tumor
progression. Reestablishment of autocrine TGF
responsiveness leading
to decreased tumorigenicity in cells with deficient TGF
receptor
function was achieved by stable transfection of RII in breast cancer
MCF-7 L cells (6) and in the human colon carcinoma cell line HCT116
(8). Based on these studies, agents that can control the expression of
TGF
receptors may have therapeutic implications. In particular,
agents that can enhance the expression level of RII in cells where it appears to be repressed may be an effective chemopreventive approach. To date, no such agents have been carefully and fully characterized for
their ability to induce RII and subsequently enhance autocrine-negative TGF
activity. Characterization of these agents should lead to a
better understanding of TGF
-mediated growth inhibition and its
anti-tumor effects.
In the present study, we demonstrated for the first time that an
increase in autocrine TGF function by the active metabolite of
vitamin D3 is solely due to an increase in RII expression. However, the effective dose of active vitamin D3, which
induces negative autocrine TGF
activity would also cause
hypercalcemia, leading to unwanted side effects. To overcome this
problem, analogues such as EB1089 have been developed that have
increased potency and reduced hypercalcemic effects (39). We have shown
here that the analogue EB1089 has similar effects to the parental
compound at lower concentrations, which make it an attractive and
potential chemopreventive agent. Thus, vitamin D3 and its
analogues can inhibit malignant cell growth through a novel mechanism
of induction of negative autocrine TGF
activity.
A number of studies have reported that expression of RI and RII protein
can be regulated by factors such as cell density (40, 41); exposure to
parathyroid, adrenal, or androgenic hormones (42-44); and TGF (45).
However, these agents do not readily lend themselves to chemopreventive
approaches. Moreover, these studies were restricted to cell surface
analysis utilizing 125I-TGF
in receptor cross-linking
and did not determine biological effects with respect to potential
autocrine activity changes. In this study, we demonstrated a 3-fold
increase in steady state RII mRNA levels (Fig. 4) by vitamin
D3 treatment, which correlated with a 3-4-fold increase in
cell surface RII protein (Figs. 5 and 6). This suggests that
translational modifications were unlikely to be responsible for
up-regulation of the RII protein. Induction of RII mRNA by vitamin
D3 may involve transcriptional and post-transcriptional mechanisms. Vitamin D3 association with VDR can either
increase the affinity of VDR binding to its target DNA sequence or
cause conformational changes in the receptor leading to alterations in
gene activation (46). VDR proteins were detected by Western analysis in
both MCF-7 E and MCF-7 L cells (data not shown). The VD-VDR complex in
combination with other steroid receptors could be directly involved in
stimulation of the RII promoter activity or act indirectly by
increasing the quantity or activity of related transcriptional
activators. Examination of the recently characterized RII promoter
region (47) did not reveal sequences analogous to the well established
vitamin D3-responsive elements, indicating that induction
of RII mRNA in MCF-7 E cells by vitamin D3 was unlikely
to be a direct effect. The lack of RII induction in MCF-7 L cells
suggests that the RII gene might be suppressed by factors or mechanisms
that were not present in MCF-7 E cells and that certain transcriptional
factors that were essential to activation of the VDR signaling pathway
might be deficient in MCF-7 L cells. Unraveling these mechanisms may
lead to novel approaches for reactivation of the RII tumor suppressor
gene.
Modulation of TGF expression or secretion by vitamin D3
has been shown in keratinocytes, chondrocytes, rat prostatic epithelial cells, and one human breast cancer cell line BT-20 (48-51). Danielpour (51) was able to demonstrate in a nontumorigenic rat prostate epithelial cell line that induction of TGF
autocrine activity by
vitamin D3 was mediated by increases in all three isoforms of TGF
. In addition, other steroid hormones have been shown to increase activation of latent TGF
while not affecting total levels (52, 53). Interestingly, modulation of TGF
levels or its activation
by vitamin D3 compounds was not observed in this study of
this strain of human breast cancer cell line (MCF-7 E). A potential difficulty with chemopreventive approaches involving TGF
ligand induction rather than receptor induction resides in the tumor-enhancing effects associated with TGF
overexpression, such as angiogenesis and
immunosuppression (54). However, EB1089 may offer an advantage in that
induction of autocrine-negative TGF
activity occurs through RII and
not its TGF
ligand. Enhancement of autocrine-negative TGF
activity, without the increase in TGF
ligand and the potential tumor-enhancing effects associated with it, makes the use of these compounds an attractive approach by offering a potential novel mechanism for cancer prevention and/or therapy.
In addition to vitamin D3, other related members of the
steroid hormone family have been shown to modulate TGF receptor
expression. In human neuroblastoma cells, RA increased RII mRNA
levels and RI protein as well as increasing expression and secretion of
TGF
1 (55). However, cell surface RII was undetectable by
receptor cross-linking in this study. The up-regulation of
TGF
1 and the TGF
receptors occurred only in the
neuroblastoma cell line that was responsive to RA-induced growth
arrest. RA treatment of RL human B lymphoma cells induced a 2-fold
increase in total RII protein. However, the study failed to examine
whether that correlated to increased cell surface RII (54). In addition
to RA, vitamin E succinate, which demonstrated potent growth
inhibition, induced RI, RII, and TGF
proteins but did not affect
their mRNA levels in RL human B lymphoma cells (56). Treatment of
the lymphoma cells with TGF
1-neutralizing antibodies
could partially block the growth inhibitory functions of vitamin E
succinate and RA, indicating that treatment induced a TGF
autocrine-negative loop. However, this study did not examine cell
surface receptor levels; thus, the increase in negative TGF
autocrine activity could be due to increased ligand levels. Both of
these studies demonstrated the ability of other agents to induce RII
mRNA or protein levels but failed to either detect or investigate
RII cell surface levels. In addition, these compounds also enhance
ligand expression, which may have adverse effects on surrounding
tissue. The observation that vitamin D3 induced autocrine
negative TGF
activity through increased cell surface RII and not
TGF
ligand may prove to be of significance in breast cancer therapy
and/or prevention.
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ACKNOWLEDGEMENT |
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We thank Mu-En Lee for kindly providing the cyclin A luciferase reporter construct.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA38173, CA50457, and CA72001 (to M. G. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to the work.
** To whom correspondence should be addressed. Tel.: 210-567-4524; Fax: 210-567-3447.
1
The abbreviations used are: TGF, transforming
growth factor-
; RA, retinoic acid; VDR, vitamin D receptor; RI, RII,
and RIII, TGF
receptor type I, II, and III, respectively.
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REFERENCES |
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