| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Priming Up-Regulates IFN-Stimulated Gene Factor 3 (ISGF3) Components, Augmenting Responsiveness of IFN-Resistant Melanoma Cells to Type I IFNs
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß, STAT2, and p48-ISGF3
, are tyrosine
phosphorylated before their assembly into a complex. Subsequently, the
ISGF3 complex is translocated to the nucleus. We have recently
established that the responsiveness of human melanoma cell lines to
type I IFNs correlates directly with their intracellular levels of
ISGF3 components, particularly STAT1. In the present study, we show
that pretreating IFN-resistant melanoma cell lines with IFN-
(IFN- priming) before stimulation with type I IFN also results in
increased levels of ISGF3 components and enhanced DNA-binding
activation of ISGF3. In addition, IFN- priming of IFN-resistant
melanoma cell lines increased expression of type I IFN-induced ISG
products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I,
B7-1, and ICAM-1 Ags. Furthermore, IFN- priming enhanced the
antiviral effect of IFN-ß on the IFN-resistant melanoma cell line,
MM96. These results support a role for IFN- priming in up-regulating
ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma
cell lines to type I IFN and providing a molecular basis and
justification for using sequential IFN therapy, as proposed by others,
to enhance the use of IFNs in the treatment of melanoma. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, a single 48-kDa DNA-binding protein; and
ISGF3, which consists of two STAT proteins, STAT2 (113 kDa) and
either STAT1 (91 kDa) or STAT1ß (84 kDa). Treatment of cells with
type I IFNs leads to association and activation by tyrosine
phosphorylation of the STAT components of ISGF3, which then complex
with p48-ISGF3 to form functional ISGF3. This complex then
translocates to the nucleus and binds to the IFN-stimulated response
element (ISRE) to activate transcription of ISGs. By contrast, the
binding of IFN- (type II IFN) to its receptor (8) stimulates only
the tyrosine phosphorylation of STAT1ß (9, 10), leading to the
formation of -activating factor (GAF), a dimer of STAT1 that forms
via SH2 domain interaction (11). GAF binds a distinct DNA sequence
element, -activating sequence (GAS), for gene induction.
Among the cytokines, IFNs were the first to be used in clinical trials
for cancer treatment. Their inhibitory effect on tumor cell growth in
vitro has been described for various cell types, including malignant
melanoma (reviewed in Refs. 12 and 13). Many clinical trials have
documented the antitumor activity of IFNs, mostly using type I
IFN-ß administered to melanoma patients during therapy (see review
in 14 . Maximal overall response rates of approximately 20 to 25%
have been reported (14). Our on-going studies of the IFN responsiveness
of human melanoma cell lines have established a correlation between the
levels of IFN-induced tyrosine-phosphorylated cellular proteins and the
responsiveness of cells to type I IFNs (15, 16). Recently, we have
examined the levels and activation of the components of the JAK-STAT
pathway of IFN-induced signal transduction in human melanoma cells,
which range from IFN sensitive to IFN resistant (16). We have found
that the IFN-resistant melanoma cells contain reduced levels of the
ISGF3 components, particularly STAT1, indicating that the
nonresponsiveness of melanoma cell lines to type I IFN may be due to a
deficiency in the expression of ISGF3 components.
Previous studies by Levy et al. demonstrated that combined treatment of
cells with IFN- for 16 to 18h followed by IFN- resulted in a
10-fold increase in activation of ISGF3 compared with cells stimulated
with IFN- alone (17, 18). The effect obtained by pretreating cells
with IFN- in this manner to enhance signal transduction of IFN-
has become known as " priming" (17). Several other studies have
also confirmed the observation that enhanced ISGF3 activation by type I
IFNs results after IFN- priming of cells (19, 20, 21, 22).
In this study, we show that IFN- priming of cultures of
IFN-resistant melanoma cell lines before adding type I IFNs results in
increased levels of ISGF3 components, particularly STAT1, and enhanced
formation of activated ISGF3 DNA-binding complexes. As a consequence,
expression of a number of ISG products in such IFN-resistant melanoma
cell lines—including ISG54, 2'-5'oligoadenylate synthase (OAS), HLA
class I, B7-1, and ICAM-1 molecules—is increased, as is also the
antiviral effect obtained with IFN-ß.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Melanoma cell lines SK-MEL-28, SK-MEL-3 (American type Culture
Collection, Manassas, VA), and MM96 (23) were grown in RPMI 1640 medium
supplemented with 10% inactivated FCS. In all experiments, melanoma
cells were stimulated with either 1000 IU/ml IFN-2a (Hoffmann-La
Roche, Basel, Switzerland) or IFN-ß (Berlex Biosciences, San
Francisco, CA) for the indicated time periods. For IFN- priming,
cells were pretreated with 1000 IU/ml IFN- (Hoffmann-La Roche) for
16 h before stimulation with IFN-2a or IFN-ß.
Preparation of total cell lysates
Cells (1 x 106 cells) were washed with ice cold 1 x PBS and lysed by the addition of 1 ml of ice cold 1x-modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 0.1% NP40, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) (24).
Antibodies
Primary Abs used in these experiments included anti-STAT2
antiserum (Signal Transduction Laboratory, Lexington, KY),
anti-STAT1 supershift, and anti-p48-ISGF3 supershift
antisera (Santa Cruz Biotechnology, Santa Cruz, CA), B7-1 antiserum
(Serotec, Oxford, U.K.), anti-OAS antiserum (gift from Prof. Gianni
Garotta, Hoffmann-La Roche), ASH1620 mouse mAb to human monomorphic
class I HLA-A, -B, -C (gift from Dr. Mauro Sandrin, Austin Research
Institute, Heidelberg, Victoria, Australia), and anti-ICAM-1
antiserum (1H4, mouse mAb to human ICAM-1, gift from Dr. Andrew Boyd,
Queensland Institute of Medical Research, Brisbane, Australia (25)).
Rabbit anti-human ISG54 serum was prepared using a purified
recombinant GST-ISG54 fusion protein including the ISG54 amino acid
sequence from residue 207–400 (26). All secondary Abs were obtained
from Silenus, Hawthorn, Australia. Secondary Abs used included
anti-mouse IgG, FITC-conjugated and horseradish
peroxidase-conjugated sheep anti-mouse IgG, sheep anti-rabbit
IgG, and donkey anti-sheep IgG.
Immunoblotting
Samples of cell lysates (2 x 105 cells) were subjected to SDS-PAGE before transfer to Hybond-ECL membrane (Amersham, Little Chalfont, U.K.). The blot was incubated with the relevant primary Ab diluted according to the manufacturer’s instruction. After stringent washing, the blot was incubated with the relevant secondary Ab and developed using an enhanced chemiluminescence kit (Boehringer-Mannheim, Mannheim, Germany). Standardization of sample loadings was confirmed by stripping and reanalyzing immunoblots for comparative levels of the housekeeping proteins HSP90 and GAP6DH as previously established (16).
Densitometric analysis
HP Scan Jet IIcx/T (Hewlett Packard) densitometer calibrated using Kodak optical density standard test strip (0.0–2.0 U) was used to scan autoradiographic film after exposure to the immunoblot chemiluminescence signal and development. The MCID-M4 (version 3, rev. 1.3) microcomputer optical imaging system (Imaging Research, St. Catherines, Ontario, Canada) was used for quantitation. The autocontouring function was applied to quantitate the relative densities of bands. The OD value for each band obtained was background subtracted before comparisons of the relative ratios of ODs were determined.
RNA preparation and Northern blotting
Total cytoplasmic RNA (20 µg) was isolated from the melanoma
cell lines by the procedure of Gough (27) and subjected to Northern
blot analysis (28). The blot was probed with an
[-32P]-labeled full-length STAT1ß cDNA (gift
of Dr. Chris Schindler, Department of Medicine, College of Physicians
and Surgeons, Columbia University, New York, NY) prepared using a
Gigaprime random priming kit (Bresatec, Adelaide, Australia). The blot
was stripped of probe by incubating the membrane in distilled water at
95°C for 30 min and subsequently reprobed with an
[-32P]-labeled PstI fragment of a rat
GAP6DH cDNA as previously described (29).
Electrophoretic gel mobility shift assay (EMSA)
Cells were incubated with IFNs as indicated. NaF (10 mM) was
added with the IFN-2a before cell lysis, to inhibit the
translocation of ISGF3 factor to the nucleus (17). Cell extracts
(1 x 107/ml cells) were prepared by resuspension
of washed cell pellets in hypotonic buffer (10 mM Tris pH 7.4, 10 mM
NaCl, 6 mM MgCl2, 10 mM KCl, 0.1 mM EGTA, 0.2 mM EDTA, 0.1
mM ZnCl2, 10 mM NaF, 0.5 mM DTT, 0.1% Nonidet P-40, 10%
glycerol, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin,
and 10 mg/ml leupeptin) followed by Dounce homogenization for 30
strokes. Cytoplasmic extracts were collected as supernatants following
a 5-min centrifugation in a microcentrifuge. Cytoplasmic extracts (10
µl) were incubated with 2 ng of [-32P]-labeled
double-stranded oligonucleotide probe corresponding to the ISRE of
ISG15 (5'-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3') and its
complement (gift of Dr. David Levy, Department of Pathology, New York
University, New York, NY) in the presence of 2.5% CHAPS
(3-[(3-cholamidopropyl) dimethylammonio]-propanesulfonate) detergent
(30) and 4 µg of double-stranded poly(dI/dC) in 1x binding buffer
(40 mM KCl, 20 mM HEPES, pH 7.4, 1 mM MgCl2, 0.1 mM EGTA,
4% Ficoll, 0.5 mM DTT, and 0.02% Nonidet P-40) in a total volume of
30 µl for 20 min at room temperature. For supershifting, p48-ISGF3
supershift Ab (1 µg) was added into the reaction mixture after 10
min, and the incubation was continued for a further 10 min. Following
incubation, 2 µl of loading dye (containing 30% glycerol, 0.25%
bromophenol blue, and 0.25% xylene cyanol) was added to each binding
reaction, and samples were then electrophoresed on a 15-cm 4%
polyacrylamide gel at 270–300 V in 0.25x TBE (0.089 M Tris-HCl, pH 8,
0.089 M boric acid, and 2 mM EDTA) at 4°C for 4 to 5 h. Gels
were dried and subjected to autoradiography.
FACS analysis
After IFN treatment, cells (3 x 105) were incubated with an appropriate dilution (in 1x PBS containing 0.5% FCS) of anti-human polymorphic class I HLA sera for 1 h on ice. After washing three times with 1x PBS, 0.5% FCS, secondary anti-mouse IgG FITC-conjugated antiserum was added, and cells were incubated for an additional hour on ice. The cells were again washed three times and incubated in fixative solution (1% formaldehyde, 0.03% NaN3, and 2% glucose) overnight before analysis by flow cytometry using a FACS IV (Becton Dickinson, Mountain View, CA). Mean fluorescence intensities of samples were recorded after gating out background fluorescence values obtained by analyzing samples stained with secondary Ab or primary Ab alone.
Antiviral and antiproliferative assays
Antiproliferative (31) and antiviral assays (32) were performed as previously described.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
priming on the expression levels of ISGF3
components STAT1, STAT2, and p48-ISGF3 in melanoma cell lines having
different IFN responsiveness
Although other pathways of signaling are activated by the IFNs,
type I IFNs predominantly transduce signals by the pathway involving
activation of the transcription factor complex ISGF3 composed of STAT1,
STAT2, and p48-ISGF3 (reviewed in Refs. 5–7). IFN- priming has
been shown previously to increase the expression of each of these ISGF3
components (18, 19, 22). Thus, the melanoma cell lines SK-MEL-28
(IFN-sensitive), SK-MEL-3 (moderately IFN-resistant), and MM96
(IFN-resistant) (15, 16, 31, 32) were examined to determine whether the
pathways involved in IFN- priming were functional, and if so,
whether it was possible to increase the levels of STAT1, STAT2, and
p48-ISGF3, thereby providing the potential for increasing the
ability of the IFN-resistant melanoma cells to respond to type I IFNs.
Thus, the three melanoma cell lines were grown in the presence and
absence of 1000 IU/ml of IFN- for 16 h. The relative levels of
STAT1 detected by immunoblotting in the different melanoma cell lines
were determined by analyzing cell lysates prepared from equal numbers
of cells (Fig. 1
B).
Controls for equal amounts of cellular protein loaded in each sample
are not shown. We routinely check that samples prepared from melanoma
cell lysates contain equivalent amounts of cellular protein by the
comparative levels of housekeeping proteins HSP90 and GAP6DH, as
previously established (16). The results from a comparison of STAT1
levels in untreated cell populations revealed that SK-MEL-28 cells
showed the highest level of STAT1, SK-MEL-3 an intermediate level, and
MM96 the lowest level. Pretreatment of cells by IFN- priming
markedly increased the levels of STAT1 detected in both the
IFN-sensitive and IFN-resistant cell lines. Quantitation by
densitometric analysis revealed that IFN- priming up-regulated the
level of STAT1 protein detected in all three melanoma cell lines by at
least eightfold (Table I). Up-regulated
STAT1 protein expression in the melanoma cell lines after IFN-
priming was consistently observed and the data presented in Figure 1B and in Table I are representative of eight different
experiments.
|
|
) revealed an increase in STAT1 mRNA
expression, occurring most noticeably in the IFN--primed SK-MEL-28
and SK-MEL-3 melanoma cells and, to a lesser extent, in the
IFN-resistant MM96 cell line. For each of the three cell lines, the
IFN-induced increase in STAT1 mRNA quantitated relative to the levels
of GAP6DH mRNA expression was determined (Table II). The results indicate that
differences occur in the manner in which the level of STAT1 expression
is regulated within the three different melanoma cell lines, with STAT1
expression in the MM96 cell line possibly occurring at the
posttranscriptional level, as the STAT1 mRNA levels in this cell line
did not change noticeably after IFN treatment, although STAT1 protein
levels markedly increased (Fig. 2).
|
|
priming has a similar up-regulatory effect on the
expression of STAT2 was also examined by immunoblotting of cell protein
extracts (Fig. 1
A). The relative level of expression
of STAT2 detected in the control nontreated cell lines was
SK-MEL-28 > SK-MEL-3 > MM96, correlating with the
responsiveness of these cell lines to IFN obtained previously (16).
IFN- priming of SK-MEL-28, SK-MEL-3, and MM96 cell lines resulted in
STAT2 expression increasing by between 4- and 5-fold, as determined by
densitometric analysis of immunoblots (Table I). These data are
representative of the results obtained from six different experiments.
The level of p48-ISGF3 also correlated with cellular responsiveness
to IFN (Fig. 1C). Thus, IFN-resistant SK-MEL-3 and
MM96 cells express less p48-ISGF3 than IFN-sensitive SK-MEL-28
cells. IFN- priming markedly increased the levels of p48-ISGF3 in
all three cell lines (by 
7- to 10-fold according to the
densitometric analysis) with the largest relative increase over control
levels occurring in the MM96 cells (Table I). Similar results were
obtained in five different experiments.
IFN- priming enhances activation of ISGF3
To analyze the effect of IFN- priming on ISGF3 activation in
the IFN-resistant melanoma cell lines, EMSAs were conducted. Activation
of ISGF3 was detected using the well-characterized interaction of
ISGF3 with the ISG15 response element (17). After IFN-2a stimulation
of IFN--primed melanoma cell lines SK-MEL-28, SK-MEL-3, and
MM96 (Fig. 3), cell lysates were prepared
and subjected to EMSA. SK-MEL-28 cells contained low levels of
activated ISGF3 when treated with IFN- or IFN-2a alone Fig. 3). However, the amount of ISGF3 binding to the ISG15
oligonucleotide probe was significantly greater in SK-MEL-28 cells that
had been IFN- primed before incubation with IFN-2a (Fig. 3). The
IFN-resistant cell lines SK-MEL-3 or MM96, when stimulated with
IFN-2a alone, contained negligible activated ISGF3 compared with
IFN-sensitive SK-MEL-28 cells (Fig. 3). However, the level of ISGF3 was
increased by IFN- priming in SK-MEL-3 and MM96 such that activated
ISGF3 became clearly detectable, although two- and fourfold greater
amounts of cell extract, respectively, had to be analyzed from these
two cell lines to detect ISGF3-DNA binding (Fig. 3).
|
priming on the responsiveness of
IFN-resistant melanoma cell lines to IFN-2a
The results from our analysis of IFN-resistant melanoma cell lines
revealed deficiencies in the expression of all of the components of
ISGF3, that is STAT1, STAT2, and p48-ISGF3, compared with their
levels detected in IFN-sensitive cell lines. In addition, levels of
expression of the ISGF3 components could be significantly increased in
both IFN-sensitive and IFN-resistant melanoma lines by priming with
IFN-. Thus, it was important to examine whether IFN- priming
could also enhance the biologic responsiveness of IFN-resistant
melanoma cell lines to type I IFNs as indicated by the production
of ISGs.
First, we analyzed the effect of IFN- priming on the IFN-induced
expression of several well-characterized ISG products, some of which
are known to be involved in mediating cellular effects of the
IFNs.
1) OAS expression is a recognized marker for IFN-induced gene
expression by either type I or II IFNs (33, 34, 35). Although either
IFN- priming for 16 h or IFN-2a treatment for 10 h
increased the levels of OAS protein in the more IFN-responsive
SK-MEL-28 and SK-MEL-3 cells (Fig. 4), we
did not observe any additional effect obtained by IFN- priming
before IFN-2a stimulation in these cells. The results indicate that
IFN-inducible OAS gene expression can be maximally activated in
SK-MEL-28 and SK-MEL-3 cells by IFN-2a alone, perhaps due to the
higher baseline expression of ISGF3 components present in the more
IFN-responsive cell lines, and thus, in this context, IFN- priming
made no difference. However, analysis of the IFN-induced expression of
OAS in the IFN-resistant MM96 cell line revealed that although OAS
levels were increased in either IFN--primed or IFN-2a-treated
cells, the level of OAS protein expression was significantly enhanced
as a result of IFN- priming before treatment with IFN-2a
(Fig. 4).
|
-primed melanoma cell
lines (Fig. 5). In all three cell lines,
greater induction of ISG54 expression was observed in IFN--primed
cells treated with IFN-ß (Fig. 5). ISG54 induction was barely
detected in SK-MEL3 and MM96 cells stimulated with either IFN-ß or
IFN- alone. However, IFN- priming of these cells greatly enhanced
the resulting expression of ISG54 induced with IFN-ß.
|
-primed melanoma cell lines on the expression of three
IFN-regulated cell surface markers known to be important to tumor cell
immunity including HLA class I (36), B7-1, and ICAM-1 (CD54) (37, 38).
A time course of treatment with either IFN- or IFN-2aß alone,
or using IFN- priming before IFN-2aß stimulation, was
conducted, and the resulting cells were analyzed by immunofluorescence
cytometry. The aim was to determine whether IFN- priming before
treatment with IFN-2a or -ß resulted in a greater increase in the
levels of expression of these IFN-regulated cell surface markers than
treatment with type I IFNs alone. In addition, we aimed to determine
the effects of varying the time course of IFN treatment and delineate
those conditions that produce the greatest simultaneous expression of
these three key cell surface markers as follows.
3) HLA class I molecules are essential for Ag presentation to activate
CD8+ cells during the induction of T cell immunity,
and cell surface expression of HLA class I Ags is known to be increased
by treatment with either type I or type II IFN (36). We examined the
effect of IFN- priming on the IFN-2a-induced level of expression
of HLA class I molecules detected by immunofluorescence staining with
Ab to monomorphic HLA class I Ag (Fig. 6). The results are summarized in Table III as mean immunofluorescence
intensities. Exposure of each of the cell lines, SK-MEL-28, SK-MEL-3,
and MM96, to either IFN- or IFN-2a increased HLA class I
expression. In all three cell lines, the induction of HLA class I
expression by IFN- was greater than that induced by IFN-2a alone.
Nevertheless, IFN- priming before IFN-2a stimulation resulted in
the highest levels of surface HLA class I expression (40–50%
higher in some cases) indicating that IFN- priming followed by
IFN-2a stimulation enhances the level of induction of HLA class
I.
|
|
and -ß were studied by immunofluorescence
analysis of the IFN-resistant melanoma cells (Tables
IV and V).
Use of either IFN- or IFN-ß alone increased expression of B7-1
molecules on all three cell lines (Table IV). However, IFN- priming
before IFN-ß stimulation resulted in much greater up-regulation of
B7-1 expression. All three cell lines showed a greater increase in B7-1
surface expression when cells were IFN- primed and subsequently
IFN-ß treated for 24 h, compared with cells treated with only
IFN-ß alone for 24 h. A larger percentage of cells (see the
gated values) showed higher levels of surface expression, especially
when cells were IFN- primed and IFN-ß treated for 48 h in
comparison with stimulation with IFN- or -ß alone for 48
h.
|
or
-ß treatment in all three cell lines, with IFN- showing the
greater effect. Cells that were IFN- primed and treated with IFN-ß
for 24 h did not show significant increases in ICAM-1 expression
compared with cells treated with IFN- alone. However, the two cell
lines, SK-MEL-28 and SK-MEL-3, which had the greater IFN
responsiveness, exhibited significantly enhanced ICAM-1 expression
after IFN- priming and treatment with IFN-ß for 48 h (Table V). By comparison, results from the IFN-resistant MM96 cell line,
examined for the relative effects of IFN- alone vs IFN- priming
before type I IFN treatment, were not as marked. The results showed
that only a slight further increase in the expression of two of the
surface markers, HLA class I and B7-1, could be obtained by treating
cells with IFN- priming for 16 h before treating them with
IFN-ß for a further 24 h, compared with the levels produced by
treatment with IFN- alone for 40 h (Tables III–V).
|
priming could enhance the biologic
responsiveness of IFN-resistant melanoma cell lines to type I IFNs as
indicated by the IFN-ß-induced antiviral response. For this study,
the IFN-resistant cell line MM96 was used. MM96 cells were IFN-
primed using 100 IU/ml of IFN-, as this concentration of IFN-
does not, by itself, have any antiviral effect on these cells (32). The
concentration of IFN required for 50% reduction in cytopathic effect
(CPE50) was reduced from 300 IU/ml (IFN-ß treated
only) to 25 IU/ml (IFN- primed and IFN-ß treated) (Fig. 7). This result clearly demonstrates that
IFN- priming potentiates the responsiveness of MM96 cells to IFN-ß
treatment.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
priming, which is known to up-regulate the
content of ISGF3 in various cell types, is shown to act in a similar
manner to increase the responsiveness of IFN-resistant cell lines to
type I IFNs.
Analysis of melanoma cells by immunoblotting of cellular proteins with
Abs to STAT1, STAT2, and p48-ISGF3 showed that IFN- priming
significantly increased expression of ISGF3 subunits, particularly
STAT1 and p48-ISGF3 (Figs. 1 and 2). The observed difference between
the immunoblot result of a highly elevated STAT1 protein expression
occurring in IFN--primed MM96 cells and less significant
IFN--induced increase in STAT1 mRNA levels detected by Northern blot
analysis in these cells indicates that a posttranscriptional mechanism
of action of IFN- priming may exist that acts to increase STAT1
protein levels in the IFN-resistant MM96 cell line. Additional support
for greater induction of activated ISGF3 in melanoma cells as a result
of IFN- priming before IFN- stimulation than the response
obtained by treatment with either IFN alone was revealed by analysis of
ISGF3-DNA binding by EMSA (Fig. 3). Our results are consistent with
those of Gao et al. (19) who demonstrated that IFN- priming
increases not only the level but also the rate of ISGF3-DNA-binding
activity following IFN- treatment. Our results also support their
contention that the increased quantities of ISGF3 that accumulate as a
result of IFN- priming are available for immediate activation by
IFN-.
Other studies have reported the ability of IFN- priming to
reconstitute the responsiveness of various cell types in vitro to
IFN-ß. For example, in the human monocytic cell line U937, which
has an undetectable basal level of ISGF3, IFN- priming has been
demonstrated to lead to accumulation of p48-ISGF3 and to potentiate
the IFN- induction of ISG expression (20). In addition, Levy et al.
(18) have shown that the combined treatment of HeLa S3 cells with
IFN- and IFN- resulted in an 10-fold increase in ISG
transcription, in comparison with cells that were stimulated by IFN-
alone; this effect occurred through an induced synthesis of
p48-ISGF3. Pretreatment of chronic lymphocytic leukemia cells with
IFN-, or the addition of extracts from IFN--treated cells,
reconstituted the level of cellular ISGF3 activity through an increase
in the level of the p48-ISGF3 component (21). In addition, retinoic
acid treatment has been shown to sensitize IFN-resistant breast cancer
cell lines deficient in STAT1 protein and promyelocytic leukemic cells
lacking p48-ISGF3 via the up-regulation of these ISGF3
components (39, 40).
One measure of the IFN responsiveness of cells is the IFN-induced
expression of ISGs. In other studies, it has been reported that where
STAT1 has been up-regulated by factors such as retinoic acid (39, 40)
or by transfection of the STAT1 gene into IFN resistant cells (16), it
is possible to restore the ISG induction by IFNs. In the present study,
we demonstrate that IFN- priming of IFN-resistant cells before
treatment with IFN-ß results in the up-regulated expression of OAS
and ISG54 (Figs. 4 and 5). This result is in agreement with other
studies showing that IFN- priming can produce a synergistic effect
through the activation of transcription of the OAS gene (33, 34, 35, 41).
The inability of IFN- priming to increase the level of OAS in the
more IFN-sensitive cell lines, such as SK-MEL-28 or SK-MEL-3, may
reflect the high basal level of OAS transcription in these cell types,
which most probably already contain sufficient levels of ISGF3 for
maximal IFN-induced OAS gene expression. However, the induction of OAS
after IFN- priming, followed by IFN- treatment, was significant
in the IFN-resistant cell line, MM96 (Fig. 4). It has been reported
that the potentiating effect of IFN- priming on ISGF3 activity and
induction of ISG transcription is less obvious in the IFN-responsive
diploid human fibroblast cell line, FS2, which has high constitutive
levels of p48-ISGF3 (18).
Results from an immunofluorescence staining analysis (Tables III–V)
also showed that IFN- priming enhanced type I IFN induction of cell
surface expression of HLA class I, B7-1, and ICAM-1 Ags. Importantly,
for tumor rejection to occur, Ags must be presented on HLA class I
molecules of melanoma cells to be recognized by CD8+
CTL (reviewed in 42 . Ag (in the form of a processed peptide)
needs to be presented via HLA class I molecules to serve as a target
for CD8+ CTL activation, and thus loss of HLA class I
expression is believed to provide an escape from immunologic control.
Tumors expressing low or nondetectable levels of HLA class I can
sometimes be rendered immunogenic by transfection of these cells with
appropriate HLA genes (43, 44, 45, 46).
Other possible reasons for poor immunogenicity of tumors may be, in
large part, a consequence of failure to express costimulatory ligands
necessary for activating CTLs. Several groups have demonstrated that
tumor immunity can be enhanced by the provision of costimulatory
signals, including B7-1 and ICAM-1 expressed on tumor cells (37, 38).
The first reports to document the effectiveness of B7-1-transfected
tumor cells as immunogens derived from studies using the murine
melanoma cell line K1735 (47, 48, 49). Recent work has established that
B7-1+ tumor cells not only induce protective immunity to
subsequent challenge with the parental tumor, but also result in
elimination of preexisting tumor (49, 50). Low levels of B7-1 molecules
have previously been reported to be present on the surface of cells
from 3 of 10 human melanomas (51), and our results extend these studies
to show that B7-1 expression levels can be enhanced by IFN- priming
before IFN- stimulation. The effect of ICAM-1 expression on
antitumor immunization has previously been shown to result in increased
rejection of murine melanoma cells (52). As with B7-1, we also show
that IFN- priming before IFN- treatment increases ICAM-1
expression on human melanoma cell lines in vitro. On balance, based on
the results with IFN-sensitive and IFN-resistant melanoma cell lines
reported here, it is indicated that treatment with IFN- priming
before treatment with type I IFNs is likely to be a more effective
strategy than therapy with type I IFNs alone, ensuring that the tumor
cells respond appropriately to the IFNs. Thus, we have demonstrated a
suitable approach, applicable to any given melanoma cell line, whereby
using IFN- priming before treatment with IFN-ß provides a basis
for achieving an optimal cellular responsiveness to the IFNs, including
such factors as maximizing the simultaneous expression of important
cell surface markers such as HLA class I, B7-1, and ICAM-1, which are
critical for effective tumor cell immunity. It is further suggested,
based on analysis of the results on IFN-induced cell surface markers in
which the effect of the time course of application of IFN- priming
and type I IFN treatment was investigated, that the timing is also
important and is a factor that will likely vary between cell lines. For
the majority of the assays conducted, treating cells by IFN- priming
for 16 h before IFN-ß treatment for a further 24 h
provided the best results (Tables III–V); longer time courses, up to
72 to 96 h, have revealed markedly diminished levels of response
(results not shown). This information will help provide greater
understanding in further attempts to improve protocols for cancer
immunotherapy using the IFNs.
One would have expected that if a deficiency in ISGF3 is the cause of
poor melanoma cell responsiveness to IFNs, then increasing the levels
of ISGF3 subunits by IFN- priming should improve the responsiveness
of resistant cells to IFN. Accordingly, IFN- priming of MM96
IFN-resistant melanoma cells before IFN-ß treatment was found to
potentiate the antiviral activity of IFN-ß on this melanoma cell line
(Fig. 7). The preceding finding is consistent with results from other
studies (20, 53, 54, 55, 56) reporting that IFN- potentiates the antiviral
actions of type I IFNs, although this is the first study to show that
an augmented IFN responsiveness can be obtained with IFN-resistant
human melanoma cell lines.
Our observations are particularly relevant to the application of the
IFNs in the therapy of human melanoma. Unfortunately, there have been
very few reports examining the effects of sequential applications of
type I and II IFNs in clinical trials of human cancers (57, 58, 59). Only
two studies examining the effects of IFN- priming in vivo have been
reported, and these involved analysis of renal cell carcinoma patients
(58, 59). It was concluded that although combined type I/II IFN
programs of treatment given concurrently were without any apparent
therapeutic benefit, the sequential delivery of IFN- followed by
IFN- provided improved therapeutic and immunomodulatory activity and
showed an objective response rate above the single-agent responses for
either agent alone. These results are consistent with our findings on
the effect of IFN- priming on increasing the responsiveness of
IFN-resistant human melanoma cell lines to type I IFNs in vitro.
In summary, our analysis of a range of IFN-mediated effects on human
melanoma target cells, including the expression of ISGF3 subunits,
ISGF3 activation, expression of ISG54, HLA class I, OAS, and the T cell
costimulatory markers, B7-1 and ICAM-1, is consistent with the role of
IFN- priming in enhancing the overall biologic responsiveness of
resistant melanoma cells to type I IFNs. Our results also highlight the
importance of such factors as the time course of treatment and the
additive effects obtainable by combined treatments with type I and II
IFNs. A deficiency in the level of ISGF3 available for signal
transduction by type I IFNs is likely to be a contributory factor to
nonoptimal antitumor activity of type I IFN therapy for melanoma and
for other cancer cell types as well. In conclusion, therefore, it is
likely that new therapeutic approaches aimed at up-regulating the ISGF3
content of melanoma cells, such as the use of IFN- priming, will
help to improve the clinical responses of melanomas to type I
IFNs.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: ISGF3, IFN-stimulated gene factor 3; ISG, IFN-sensitive gene; OAS, 2'5'-oligoadenylate synthetase; ISRE, IFN-stimulated response element; EMSA, electrophoretic mobility shift assay.
Received for publication October 31, 1997. Accepted for publication January 30, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induces rapid tyrosine phosphorylation of the subunit of its receptor. J. Biol. Chem. 267:2502. and -ß receptor: characterization and molecular cloning. Cell 77:391.[Medline]
receptors by sequential affinity chromatography on immobilized monoclonal anti-receptor antibodies and human interferon-. J. Exp. Med. 165:988. and - signal transduction pathway. EMBO J. 12:4221.[Medline]
signal transduction pathway by the protein tyrosine kinase JAK-2. Nature 366:166.[Medline]
. J Biol. Chem. 272:28779.-stimulated transcription, reconstituted in vitro. Genes Dev. 3:1362. and interferon- through induced synthesis of the one subunit of the transcription factor ISGF3. EMBO J. 9:1105.[Medline]
-priming effects in activation and deactivation of ISGF3 in K562 cells. J. Biol. Chem. 268:12380. potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon- in U937 cells. J. Interferon Res. 12:87.[Medline]
in vitro are defective in the activation of the DNA-binding factor interferon-stimulated gene factor 3. Blood 84:1942. and -ß: patterns of induction of 2–5 A synthetase and a dsRNA dependent protein kinase in mouse cells and human cells. J. Interferon Res. 1:191.[Medline]
, -ß and - in human cell lines differing in their sensitivity to the anticellular and antiviral activities of these interferons. Virology 117:425.[Medline]
on 2',5'-oligoadenylate synthetase induction by huIFN-. J. Interferon Res. 6:445.[Medline]
both act synergistically to produce an antiviral state in cells resistant to both Interferons individually. J. Virol. 63:4560.
, -ß and -. J. Virol. 49:490. and - or -ß. J. Interferon Res. 5:361.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. A. Gerber and J. S. Pober IFN-{alpha} Induces Transcription of Hypoxia-Inducible Factor-1{alpha} to Inhibit Proliferation of Human Endothelial Cells J. Immunol., July 15, 2008; 181(2): 1052 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. B. Lesinski, J. Trefry, M. Brasdovich, S. V. Kondadasula, K. Sackey, J. M. Zimmerer, A. R. Chaudhury, L. Yu, X. Zhang, T. R. Crespin, et al. Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-{alpha} Compared with Immune Effector Cells Clin. Cancer Res., September 1, 2007; 13(17): 5010 - 5019. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Stout, K. Melendez, J. Seagrave, M. J. Holtzman, B. Wilson, J. Xiang, and Y. Tesfaigzi STAT1 Activation Causes Translocation of Bax to the Endoplasmic Reticulum during the Resolution of Airway Mucous Cell Hyperplasia by IFN-{gamma} J. Immunol., June 15, 2007; 178(12): 8107 - 8116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. E. Buckley, A. M. Hosey, J. J. Gorski, J. W. Purcell, J. M. Mulligan, D. P. Harkin, and P. B. Mullan BRCA1 Regulates IFN-{gamma} Signaling through a Mechanism Involving the Type I IFNs Mol. Cancer Res., March 1, 2007; 5(3): 261 - 270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. Ho and L. B. Ivashkiv Role of STAT3 in Type I Interferon Responses: NEGATIVE REGULATION OF STAT1-DEPENDENT INFLAMMATORY GENE ACTIVATION J. Biol. Chem., May 19, 2006; 281(20): 14111 - 14118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Lavergne, B. Combadiere, O. Bonduelle, M. Iga, J.-L. Gao, M. Maho, A. Boissonnas, P. M. Murphy, P. Debre, and C. Combadiere Fractalkine Mediates Natural Killer-Dependent Antitumor Responses in Vivo Cancer Res., November 1, 2003; 63(21): 7468 - 7474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. H. Wong, H. Sim, M. Chatterjee-Kishore, I. Hatzinisiriou, R. J. Devenish, G. Stark, and S. J. Ralph Isolation and Characterization of a Human STAT1 Gene Regulatory Element. INDUCIBILITY BY INTERFERON (IFN) TYPES I AND II AND ROLE OF IFN REGULATORY FACTOR-1 J. Biol. Chem., May 24, 2002; 277(22): 19408 - 19417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nishio, M. Tsurudome, M. Ito, M. Kawano, H. Komada, and Y. Ito High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons J. Virol., October 1, 2001; 75(19): 9165 - 9176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, C. M. Horvath, and S. Waxman Regrowth of 5-Fluorouracil-treated Human Colon Cancer Cells Is Prevented by the Combination of Interferon {{gamma}}, Indomethacin, and Phenylbutyrate Cancer Res., June 1, 2000; 60(12): 3200 - 3206. [Abstract] [Full Text]
|
|
|
|
|
|
D. Zella, O. Barabitskaja, L. Casareto, F. Romerio, P. Secchiero, M. S. Reitz Jr., R. C. Gallo, and F. F. Weichold Recombinant IFN-{alpha} (2b) Increases the Expression of Apoptosis Receptor CD95 and Chemokine Receptors CCR1 and CCR3 in Monocytoid Cells J. Immunol., September 15, 1999; 163(6): 3169 - 3175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Therapeutic Effects of Tumor-Reactive Type 1 and Type 2 CD8+ T Cell Subpopulations in Established Pulmonary Metastases J. Immunol., June 1, 1999; 162(11): 6671 - 6680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xiao, L. Wang, X. Yang, T. Chen, D. Hodge, P. F. Johnson, and W. Farrar CCAAT/Enhancer-binding Protein beta Mediates Interferon-gamma -induced p48 (ISGF3-gamma ) Gene Transcription in Human Monocytic Cells J. Biol. Chem., June 22, 2001; 276(26): 23275 - 23281. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
