From the Department of Pathology, Anatomy and Cell Biology and the
Kimmel Cancer Center, Jefferson Medical College, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Perlecan, a heparan sulfate proteoglycan of
basement membranes and cell surfaces, has been implicated in the
control of tumor cell growth and metastasis because of its ability to
bind and store growth factors and its activity as an inducer of
angiogenesis. Because interferon- (IFN-
), a cytokine with known
antiproliferative and antitumoral activity, binds with high affinity to
the heparan sulfate side chains of perlecan, we investigated the
activity of IFN-
on perlecan gene expression and cell growth in
colon carcinoma cells. We found that IFN-
rapidly and efficiently
blocked perlecan gene expression with concurrent growth
suppression, a phenomenon that was independent of a functional
p21WAF1/CIP1. These effects were transcriptionally mediated,
did not require new protein synthesis, and were fully reversible.
Moreover, we found these IFN-
-induced effects to be generalizable
because they could be reproduced in a variety of cells with various
histogenetic backgrounds. The transcriptional repression of the
perlecan gene required intact Stat1 protein, and these effects were
likely mediated by Stat1-binding sites in the distal promoter region.
Thus, the IFN-
-mediated transcriptional repression of perlecan may
represent a novel antitumoral effect of this cytokine through which it
eliminates a powerful angiogenic stimulus.
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INTRODUCTION |
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Proteoglycans play key roles during inflammation, tissue remodeling, and cancer growth primarily through their ability to act as modifiers of growth factors and by actively participating in the fine tuning of receptor/ligand interactions (1). An important member of the proteoglycan gene family is perlecan, a ubiquitous heparan sulfate proteoglycan that has been involved in the control of cell proliferation, tumor invasion, and angiogenesis (2). The complex structure of perlecan protein core (3) and its widespread distribution (4, 5) suggest that this gene product is involved in several fundamental biological processes. For instance, increased perlecan levels are found in metastatic melanomas (6) and correlate with a more aggressive behavior (7). In tumor xenografts induced by subcutaneous injection of human prostate carcinoma cells in nude mice, human-derived perlecan was deposited along the basement membrane of newly formed murine vessels (2). Thus, perlecan may act as a scaffold upon which proliferating capillaries grow and eventually form functional blood vessels (2). Indeed, perlecan binds basic fibroblast growth factor (FGF-2)1 (8, 9) and acts as a low affinity receptor for FGF-2 thereby promoting angiogenesis (10). FGF-2 binds directly to the heparan sulfate side chains located in the N-terminal Domain I, and its release by proteolytic processing (11) may represent a biological mechanism by which dynamic molecules can be functionally disengaged at the active site of tissue remodeling and tumor invasion (12). In support of this view, reduction of endogenous perlecan levels by stable antisense transfection causes a marked suppression of autocrine and paracrine function of FGF-2 in melanoma cells (13). In contrast, antisense expression of perlecan cDNA induces tumorigenesis in fibrosarcoma cells and a faster appearance of tumors in nude mice (14). Thus, the cellular context is important in mediating the functions of perlecan.
A systematic study of murine embryogenesis has revealed that perlecan expression appears early in tissues of vasculogenesis such as the heart primordium and major blood vessels (15). Subsequently, perlecan mRNA levels increase, and this correlates with the onset of tissue differentiation of various organs including kidneys, lungs, spleen, liver, and gastrointestinal tract. In adult human tissues, perlecan epitopes are distributed along all vascularized organs, including the sinusoidal spaces of the liver, spleen, and lymphoreticular organs (4). The latter suggests that perlecan may play a role in the normal development and homeostasis of lymphoid organs.
In addition to binding a variety of extracellular matrix proteins,
perlecan binds to transforming growth factor- and is
transcriptionally induced by this cytokine (16) and also binds to
interferon-
(IFN-
) (17) with high affinity (Kd = 10
9 M) (18). Interferon-
is a
glycoprotein synthesized primarily by T lymphocytes and is involved in
the regulation of the immune response (19). It possesses antiviral,
antiproliferative, and antitumoral activity in a number of cells and
triggers a signaling cascade that leads to the expression of early
response genes through the activation of the signal transducers and
activators of transcription (STAT) proteins (19, 20). This activation
occurs through both Tyr phosphorylation, which is critical for the
translocation and binding to the DNA, and Ser/Thr phosphorylation,
which is crucial for maximal transcriptional activation (19). Upon
activation, STAT proteins dimerize and translocate into the nucleus
where they bind to the IFN-
activated site (GAS) (19).
Because one of the main goals of our laboratory is to understand how
specific cytokines regulate perlecan gene expression, we embarked on a
study to determine whether IFN- affects perlecan gene expression
and, if so, to characterize the mechanism of action. We discovered that
IFN-
concurrently suppressed tumor cell growth and perlecan gene
expression. These effects were transcriptionally mediated, did not
require new protein synthesis, were fully reversible, and were general
since they could be reproduced in a variety of cells with various
histogenetic backgrounds. The results also showed that the
transcriptional repression of the perlecan gene required a functional
Stat1 protein and that these effects were likely mediated by
Stat1-binding sites located in the distal promoter region. Thus, the
IFN-
-mediated transcriptional repression of perlecan may represent a
novel antitumoral effect of this cytokine through which it eliminates a
powerful angiogenic stimulus from the tumor microenvironment.
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EXPERIMENTAL PROCEDURES |
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Materials and Cell Cultures--
[-32P]dCTP and
[14C]chloramphenicol were obtained from Amersham Corp.
Human recombinant IFN-
was purchased from Boehringer Mannheim. HeLa
cervical carcinoma, WiDr/HT29 colon carcinoma, Saos-2 osteosarcoma, HT-1080 fibrosarcoma, and Hep G2 hepatoma cells were obtained from the
American Type Culture Collection. HCT116 p21 (+/+) and p21 (
/
)
colon carcinoma cells (21) and U3A (Stat1
/
) and 2FTGH (Stat1 +/+)
cells (22) were described before. Cells were routinely maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Life Technologies, Inc.), 2 mM glutamine, and
100 units/ml penicillin. Perlecan promoter-CAT constructs were used as
described previously (16). The cDNA HS15 was described before
(3).
Transient Cell Transfection and CAT Assays--
Transient cell
transfection was performed by the calcium phosphate procedure as
described before (16). Briefly, subconfluent cells were co-transfected
in suspension with 20 µg of perlecan promoter-CAT and 10 µg of
pSV--galactosidase plasmid to provide an internal standard for
normalization of transfection. After 48 h, the cells were washed,
incubated in Dulbecco's modified Eagle's medium for 12 h in the
presence or absence of IFN-
, and assayed for
-galactosidase
activity (16). For CAT assay, the products were resolved on preslotted
thin layer chromatography plates in a chloroform/methanol (95:5) mobile
phase and processed for autoradiography followed by laser scan
densitometry (16).
Western Immunoblotting, Cell Proliferation Assays, and
Fluorescence-activated Cell Sorter (FACS)--
For immunoblotting,
106 cells were seeded in six-well dishes and incubated in
serum-free media in the presence or absence of IFN-. Serial
dilutions of the media were applied to nitrocellulose filters
(Schleicher and Schuell) and air-dried. The membranes were blocked with
4% Carnation non-fat milk in Tris borate buffer (TBS) and incubated
with a monoclonal antibody directed against Domain III of perlecan (4).
After three washes in TBS-0.1% milk, the membranes were incubated with
an anti-mouse IgG labeled with horseradish peroxidase, washed in
TBS-0.1% milk, and chemiluminescence (NEN Life Science Products) was
detected by autoradiography for 10-60 s. To establish the growth rates
of the cells exposed to IFN-
, we used two independent approaches:
cell counting and a nonradioactive cell proliferation assay (Promega)
where the amount of formazan product is proportional to the number of
viable cells (23). FACS analysis was used to analyze the cycling
characteristics of the cell populations.
Isolation of RNA and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-- Total RNA was prepared with the TRI-ReagentTM (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer's recommendations. The RT was performed using 5 µg of total RNA (24). Briefly, the reaction was performed at 42 °C in RT buffer containing 5 mM dNTPs, 1 µM perlecan antisense primer from Domain IV (bp 9259-9237), and 1 µM GAPDH antisense primer. For PCR, 5 µl of the RT reaction was added to a mixture containing 2 mM dNTPs, PCR buffer, two perlecan primers from bp 7950-7966 (sense primer) and bp 9067-9051 (nested antisense primer) of Domain IV, and the GAPDH-specific sense primer. The PCR reaction comprised 25 cycles with 1 min at 94 °C, 2 min at 55 °C, and 3 min at 72 °C. A 25-µl aliquot of each PCR reaction was separated on a 1% agarose gel and transferred to nitrocellulose in 10× SSC. The membrane was hybridized with either a probe spanning Domain IV of perlecan (HS166) or GAPDH, washed in 0.5× SSC/0.1% SDS at 65 °C, air-dried, and exposed to x-ray films for 10-30 min.
Nuclear Run-on Transcription Assay--
Nuclear run-on was
performed as described before (25) with minor modifications. Each
reaction consisted of 210 µl of nuclei and 60 µl of 5× run-on
buffer (25 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2, 750 mM KCl, and 1.25 mM
triphosphates of A, G, and C). [-32P]UTP (30 µl,
3000 Ci/mM) was added, and the nuclear suspension was
incubated at 30 °C for 30 min, after which DNase I (5 µg/ml) was
added for 5 min. The reaction was then made 1× SET (1% SDS, 5 mM EDTA, 10 mM Tris-HCl, pH 7.4), and 200 µg/ml proteinase K was added for 45 min at 37 °C. Following
phenol/chloroform extraction and isopropyl alcohol precipitation, the
pelleted nuclei were purified further through a G-50 spin column. The
eluate was made 0.2 M in NaOH and after 10 min on ice,
HEPES was added to a concentration of 0.24 M. Following
ethanol precipitation for 18 h at
20 °C, the purified pellets
were resuspended in hybridization buffer (10 mM Tris, pH
7.4, 0.2% SDS, 10 mM EDTA, 0.3 M NaCl, 1×
Denhardt's solution, and 250 µg/ml salmon sperm DNA).
Nitrocellulose filters containing the plasmid DNAs were hybridized for
36 h at 65 °C, washed twice for 15 min in 0.1% SDS, 2× SSC at
room temperature, washed once at 60 °C (0.1% SDS, 0.1× SSC) for 30 min, and processed for autoradiography as above.
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RESULTS AND DISCUSSION |
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The Effects of IFN- on the Growth of Human Colon Carcinoma
Cells: a Block in Cell Cycle Progression That Does Not Require a
Functional p21--
In the first sets of experiments we investigated
the effects of IFN-
on the growth of the human colon carcinoma cells
WiDr/HT29 (26). These cells represent a useful model system to study
growth and proteoglycan biology because they synthesize high levels of perlecan (27) and respond to a variety of factors including transforming growth factor-
(28), nerve growth factor (6), and
phorbol ester (24). The results showed a complete growth arrest,
without any evidence of cell death, when the tumor cells were exposed
to ~9 nM IFN-
(160 ng/ml) for various periods of time
(Fig. 1A). This growth
suppression was fully reversible (Fig. 1B), and
dose-response studies performed by exposing the cells to increasing
amounts of IFN-
for 2 (Fig. 1C) or 3 (not shown) days
revealed nearly complete inhibition at 80 ng/ml, with an IC50 of ~50 ng/ml (3 nM). To determine
whether the inhibitory effects of IFN-
were due to a block in the
onset of DNA synthesis, the growth state of the cells was tested by
FACS analysis. Interferon-
increased the proportion of cells in
G1 from 50% to 73% and decreased the proportion of cells
in S and G2 phases (Fig. 1D). Because it was
previously shown that activation of Stat1 by IFN-
led to growth
suppression via induction of p21 (29), we tested HCT116 colon carcinoma
cells that harbor a targeted disruption of both p21 alleles (21). The
results clearly showed that p21 is not required for growth arrest in
these colon carcinoma cells since comparable growth suppression was
observed in HCT116 cells with either a p21 +/+ (Fig. 1E) or
p21
/
(Fig. 1G) genotype. Similarly, FACS analysis
revealed a proportional increase in the percentage of cells in
G1 phase of the cell cycle (Fig. 1, F and
H).
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IFN- Markedly Suppresses Perlecan Gene Transcription and Its
Effects Are Fully Reversible--
Because perlecan has been linked to
tumor progression by virtue of its ability to modulate angiogenic
stimuli such as FGF-2 and FGF-7 (9, 10), we tested whether the
expression of this key proteoglycan was modulated by IFN-
. We used
RT-PCR and a primer set that would amplify a perlecan-specific 1.1-kb
fragment encoding part of Domain IV (24). The analysis was performed in
the linear range thus allowing quantitative assessment of the perlecan
levels vis á vis those of the housekeeping gene
GAPDH which was concurrently amplified in the same test
tube. The levels of perlecan mRNA were markedly suppressed (Fig.
2A, lane 2), and these effects were maintained for up to 48 h (Fig. 2A,
lane 3). Moreover, the suppression of perlecan mRNA
transcript was fully reversible (Fig. 2A, lanes 4 and 5). The down-regulation of perlecan mRNA steady
state levels was corroborated by a time course analysis of perlecan
protein core synthesis by Western immunoblotting using a monoclonal
antibody directed against perlecan Domain III (Fig. 2B). The
amount of secreted perlecan following IFN-
treatment was markedly
reduced by ~80% after 2 h and by ~90% after 4 h, with a
calculated T1/2 of ~1 h. Notably, this inhibition
remained relatively constant for up to 48 h, in agreement with the
mRNA data. The slight increase at later time points (Fig.
2B) is likely due to the decline of activity of the IFN-
because we used one single bolus of the cytokine. Because the half-life
of perlecan in WiDr/HT29 colon carcinoma cells is relatively long,
~75 min (30), the inhibitory effects of IFN-
on perlecan gene
transcription would have been even higher than calculated.
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Transcriptional Suppression by IFN---
The dramatic decrease
in perlecan mRNA and protein core levels described above upon
treatment with recombinant IFN-
could be accounted for by a decrease
in transcriptional rates, a decline in mRNA stability, or by
alterations in nuclear processing of the nascent RNA molecules (or by a
combination of these processes). To measure transcription of the
perlecan gene directly, we employed nuclear run-on assay, a direct
measure of RNA polymerase density, and hence transcriptional activity
(32). Nascent RNA labeled with [32P]UTP from nuclei
isolated from control or IFN-
-treated cells was hybridized to
membranes preslotted with various pBluescript plasmids containing
perlecan, GAPDH, or insert-less vector. The results showed a marked
suppression of perlecan transcriptional activity within 2 h of
exposure to IFN-
, and these effects were maintained for 4 h
(Fig. 3A). Under the same
conditions the transcriptional rates of GAPDH were slightly increased,
also confirming the lack of cytotoxic effects of our assay.
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The Transcriptional Repression of Perlecan by IFN- Requires a
Functional Stat1 Protein--
Next, we wanted to dissect the potential
mechanism through which IFN-
may regulate perlecan gene
transcription. To this end, we determined whether the effects of
IFN-
required a functional Stat1, a transcription factor known to be
regulated by IFN-
(19). We utilized Stat1-deficient U3A cells and
the parental Stat1 +/+ 2FTGH cells. The mutant U3A cells are derived
from HT-1080 fibrosarcoma cells and are defective in their response to
IFN-
because of a lack of Stat1 (22). As predicted, the U3A cells
were insensitive to IFN-
in terms of growth suppression whereas the
parental cell line responded quite efficiently (Fig.
4A). As in the case of the
WiDr/HT29 colon carcinoma cells, the control 2FTGH cells also showed a
significant (55-60%) inhibition of perlecan protein core levels by
Western immunoblotting in contrast to the Stat1
/
U3A cells, which
were totally unresponsive (Fig. 4B). Transient cell
transfection assays using the full-length perlecan-CAT promoter construct showed significant transcriptional suppression only in the
Stat1 +/+ cells (Fig. 4C). Thus, we conclude that a
functional Stat1 protein is required to mediate the transcriptional
silencing of the perlecan gene.
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ACKNOWLEDGEMENTS |
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We thank I. Eichstetter for excellent
technical assistance and Drs. B. Vogelstein (Johns Hopkins University)
and G. Stark (Cleveland Clinic Foundation Research Institute) for
generously providing the p21 /
and Stat1
/
cell lines,
respectively.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 CA39481 and RO1 CA47282 (to R. V. I).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.
§ To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Rm. 249, JAH, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail: Iozzo{at}lac.jci.tju.edu.
1
The abbreviations used are: FGF, fibroblast
growth factor; IFN-, interferon-
; STAT, signal transducer and
activator of transcription; GAS, IFN-
-activated site; FACS,
fluorescence-activated cell sorter; p21, the
cyclin-dependent kinase inhibitor p21WAF1/CIP1;
RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; TBS, Tris borate buffer; bp, base pair(s); kb,
kilobase(s).
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REFERENCES |
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