From the Department of Immunology, Mayo Clinic/Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Interferon- (IFN-
) has been used as therapy
for the treatment of a variety of viral diseases and malignancies
including multiple myeloma. The effectiveness of interferon-
in
treating multiple myeloma, however, has been somewhat variable, and the mechanism(s) accounting for this is not well understood. As a means to
examine the basis for the differential effectiveness of this cytokine,
we have analyzed IFN-
-mediated modulation of the cell cycle in two
human myeloma cell lines. These two cell lines, ANBL-6 and KAS-6/1,
display dramatically different outcomes in response to this cytokine.
Although IFN-
inhibited the growth of ANBL-6 cells by blocking cell
cycle progression from G0/G1 to S phase,
IFN-
stimulated cell cycle progression in KAS-6/1 cells. Moreover,
the effects of IFN-
on cell cycle progression correlated with the
phosphorylation status of the retinoblastoma protein. Of interest,
IFN-
increased cyclin D2 expression and cyclin-dependent
kinase activity in the KAS-6/1 cells but not in the ANBL-6 cells. To
determine whether the differential effects of IFN-
on myeloma cell
cycle progression could also result from differences in the expression
of cyclin-dependent kinase inhibitors, we examined the
effects of IFN-
on the induction of cyclin-dependent kinase inhibitors with broad regulatory function (p21 and p27) and
those with specificity for G1-associated
cyclin-cyclin-dependent kinase complexes (p15, p16, p18,
and p19). Although we failed to detect an effect of IFN-
on
expression levels of p21, p15, p16, or p18, IFN-
treatment of the
ANBL-6 cell line resulted in induction of p19 expression, whereas it
was without effect on the KAS-6/1 cell line. These results suggest that
heterogeneity in IFN-
-mediated growth effects in myeloma cells
correlates with differential induction of cyclin D2 and
p19INK4d expression.
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INTRODUCTION |
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Interferon-alpha
(IFN-)1 is a natural
therapeutic agent that has been used to treat a variety of viral
diseases and malignancies, including multiple myeloma (MM), a B cell
malignancy characterized by the clonal expansion of plasma cells
(1-3). Although IFN-
typically has an anti-proliferative effect on
a wide variety of cell types, thereby providing the rationale for its
clinical use, IFN-
treatment of MM patients has not been universally
effective. Indeed, several reports have demonstrated that IFN-
therapy may actually result in disease aggravation and stimulation of
plasma cell growth (4, 5). Understanding the mechanisms by which some
myeloma cells are adverse to the cell cycle inhibitory effects of
IFN-
is essential to permit the effective use of IFN-
in the
treatment of MM.
Cell cycle progression is regulated by proteins called cyclins and cyclin-dependent kinases (CDKs) that associate with each other in a cell cycle phase-specific manner (reviewed in Ref. 6). The process of cyclin-dependent activation of CDKs is counterbalanced by CDK inhibitors (CKIs). The first family of CKIs, referred as the p21cip1 family, includes at least three proteins, and each member of this family binds to several cyclin-CDK complexes (7). The second family of CKIs, referred to as the INK4 family, specifically binds to CDK4 and CDK6 and hence is specific for regulation of the G1 phase (8-10). Numerous studies have demonstrated the importance of the INK4 family in overall cell growth control (11-16).
The CDK inhibitors function by inhibiting CDK-mediated phosphorylation
of the retinoblastoma protein (pRb), a known tumor suppressor (17, 18).
Thus, during G1 phase, phosphorylation of pRb is regulated
by the periodic expression of INK4 CKIs and cyclin Ds, which determines
whether active cyclin D-CDK or inactive inhibitor-CDK complexes will be
assembled. Growth arrest of hematopoietic cell lines by IFN- has
been shown to correlate with inhibition of pRb phosphorylation (19,
20). Considerable effort has been directed at the analysis of IFN-
signaling; however, the role of CKIs in mediating inhibition of pRb
hyperphosphorylation and cell cycle progression by IFN-
remains to
be elucidated. Although p27kip1 has been shown to be induced following
IFN-
treatment in some cell lines, its kinetics of expression are
not consistent with a role for this inhibitor in the regulation of
early G1 phase arrest (21). Induction of p21Cip1 expression
has also been suggested to underlie IFN-
-mediated growth arrest of
Daudi cells, and this occurred in a pRb-dependent manner
(22, 23). However, p21 expression was also induced in IFN-
growth-arrested pRb
/
U-266 cells and in
IFN-
-resistant H9 cells (22). In contrast, Yamada et al.
(21) did not observe p21 induction in Daudi cells by IFN-
treatment.
Thus, it remains unclear whether the anti-proliferative effects of
IFN-
are indeed mediated by p21 or whether IFN-
acts instead via
induction of other cell cycle regulatory protein(s). In addition, the
role of the INK4 family members has not been clearly defined in
IFN-
-mediated cell cycle regulation. Although IFN-
treatment of
the U-266 cell line was reported to increase p15 and p16 mRNA
expression, these genes were not induced in IFN-
growth-arrested
Daudi cells (22). As the U-266 cell line is pRb
/
, the
role of these CKIs in mediating the anti-proliferative effects of
IFN-
is not understood. Finally, in the same study, IFN-
had no
effect on the other two members of the INK4 family, p18 and p19
(22).
We have previously described the establishment of a panel of
interleukin-6 (IL-6) responsive human myeloma cell lines and have shown
that one of the cell lines, KAS-6/1, is uniquely growth stimulated by
IFN- (24). We have also shown that the differential growth
responsiveness of MM cell lines to IFN-
does not seem to result from
differential activation of STAT factors (24). To gain more insight into
the differential growth regulation of myeloma cells by IFN-
, in this
study we have focused on the analysis of the molecular events
regulating cell cycle progression.
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EXPERIMENTAL PROCEDURES |
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Cell Lines, Culture Medium, and Reagents--
The myeloma cell
lines ANBL-6 and KAS-6/1 were derived in our laboratory and are
maintained with IL-6 (kindly provided by Immunex Corp., Seattle, WA) as
described previously (25, 26). Recombinant purified IFN-2b was
purchased from Schering Corp. and was used at a concentration of 1000 units/ml. Monoclonal antibodies (mAb) to pRb and CDK4 as well as
polyclonal Abs to cyclin D were purchased from Upstate Biotechnology
Inc. Rabbit polyclonal antisera against the carboxyl-terminal region of
human p33CDK2 was a generous gift of Dr. R. T. Abraham (Mayo
Clinic, MN). GST-Rb fusion protein, mouse mAb to p21, and rabbit
polyclonal Abs to p15, p16, p18, and p19 were purchased from Santa Cruz
Biotechnology, Inc.
Cell Cycle Analysis--
Before analyzing the effects of IFN-
on cell cycle progression, cells were starved of IL-6 for 48 h in
medium supplemented with 0.5% bovine serum albumin. Cells were then
washed, recultured in media supplemented with bovine serum albumin at a
density of 1 × 106 per ml, and stimulated with 1 ng/ml IL-6 or 500 units/ml IFN-
. Cell cycle analysis was
subsequently performed using propidium iodide staining as described
previously (27).
Immunoprecipitation and Immunoblotting-- As described above, myeloma cells were cultured in IL-6 free media for 48 h prior to addition of the indicated cytokines. Cells (0.5-1.0 × 107) were then lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 10% glycerol, 0.1 mM EDTA, 200 mM NaCl, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin for 30 min on ice. For direct immunoblot analysis, cell lysates containing 50 µg of total protein were resolved by electrophoresis through 7.5% SDS-polyacrylamide gels and then transferred onto Immobilon-P membranes (Millipore Corp. For immunoprecipitation, cell lysates were first precleared with protein A-Sepharose (Pharmacia, Uppsala, Sweden) and then immunoprecipitated with the indicated Abs at 4 °C for 2 h. The immune complexes were collected by adsorption to protein A-Sepharose beads by overnight agitation at 4 °C. The immunoprecipitates were washed twice with lysis buffer and once with cold phosphate-buffered saline. The complexes were then boiled in 30 µl of 2 × SDS sample buffer and resolved on an SDS-PAGE gel followed by transfer to Immobilon-P membranes. The membranes were blocked (25 mM Tris-Cl (pH 7.2), 150 mM NaCl, 0.05% Tween 20, 2% bovine serum albumin) and subsequently probed with the indicated Abs. For reprobing the same blot, the membranes were stripped with 7 M guanidine, renatured, and blocked before immunoblotting with the indicated Abs. Immunoreactive proteins were detected using an enhanced chemiluminescence detection system (Amersham).
Histone H1 Kinase Assay--
Cells were lysed on ice in H1
kinase lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, 5 mM NaF, 0.1 mM Na3VO4,
1 mm DTT, and protease inhibitors) and then immunoprecipitated with
CDK2 antisera and protein A-Sepharose for 1 h at 4 °C.
Immunoprecipitates were washed twice with lysis buffer followed by two
washes with kinase buffer (20 mM HEPES, pH 7.0, 10 mM -glycerophosphate, 5 mM
MgCl2, 1 mM DTT, and 10 µg/ml leupeptin).
Kinase activity of the immunoprecipitated complex was assessed using
histone H1 as a substrate in a reaction buffer consisting of 0.6 mg/ml
histone H1 (Sigma) and 5 µCi [
-32P]ATP/reaction and
5 µM cold ATP. Following a 10-min incubation at 30 °C,
the reaction was stopped by addition of ice-cold 20 mM
EDTA, pH 8.0. Duplicate aliquots of each sample supernatant were
spotted onto phosphocellulose paper. The papers were immersed briefly
in 1% H3PO4 solution containing 10 mM Na4P2O7. After three additional 15-min washes in 1% H3PO4,
radioactivity incorporated in filter-bound histone was quantitated by
liquid scintillation counting.
CDK4 Kinase Assay--
Cells were lysed on ice in lysis buffer
(50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20, 10%
glycerol, 1 mM DTT, 10 µg/ml leupeptin, 5 µg/ml
pepstatin, 10 mM -glycerophosphate, 0.1 mM
sodium orthovanadate) and immunoprecipitated with CDK4 Abs (UBI) and
protein A-Sepharose for 2 h. Immunoprecipitates were washed twice
with lysis buffer and twice with kinase buffer (50 mM
HEPES, pH 7.5, 1 mM NaF, 2.5 mM EGTA, 1 mM DTT, 10 mM
-glycerophosphate, 0.1 mM Na3VO4, 10 mM
MgCl2). The kinase reaction was carried out for 30 min at
30 °C in a reaction buffer containing 1 µg/ml GST-pRb, 10 µCi
[
-32P]ATP/reaction, and 20 µM cold ATP.
SDS-PAGE sample buffer was added, and proteins were eluted by boiling
and resolved by 10% SDS-PAGE. The phosphorylated substrate was
detected by autoradiograph followed by immunoblotting for CDK4.
Reverse Transcriptase Polymerase Chain Reaction (PCR)--
Total
RNA was isolated using the TRIZOL reagent (Life Technologies, Inc.) and
reverse transcribed using a first strand cDNA synthesis kit
(Pharmacia). PCR reactions contained 1.5 mM
MgCl2, 1 mM dNTPs, 1 µM each of
sense (S) and antisense (AS) oligonucleotides, and 2.5 units
Taq DNA polymerase (Promega). The primer sequences and their
expected sizes are as follows: -actin,
S-5'-gacttcgagcaagagatggccac-3' and AS-5'-caatgccagggtatggtggtg-3' (265 bp); p19, S-5'-ctcacacccttggagctgg-3' and AS-gcagcctgaggcgcagaag-3'
(354 bp); p18, S-5'-ctgcaggttatgaaacttgg-3' and
AS-5'-ttattgaagatttgtggctcc-3' (382 bp) (28); p15,
S-5'-ctgcgcgtctgggggctgc-3' and AS-5'-cctcccgaaacggttgactc-3' (165 bp)
(28), p16, S-5'-gccactctcacccgacc-3' and AS-5'-ctacgaaagcggggtgg-3'
(351 bp). PCR products were visualized by electrophoresis on 2%
agarose gels and staining with ethidium bromide after 30 cycles
(denaturation at 95 °C for 1 min, annealing at 60 °C for 2 min,
and extension at 72 °C for 3 min).
Data Presentation and Analysis-- In experiments requiring visualization of bands, computer-generated images were obtained by scanning the original autoradiograms or digitalized images of ethidium bromide stained bands. In all cases, the images are representative of the original data.
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RESULTS |
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Kinetics of Myeloma Cell Line Responsiveness to IFN---
The
experiments undertaken in this study were designed to analyze
IFN-
-regulated cell cycle control in myeloma cells. The first
experiments in this study therefore analyzed the effects of
IFN-
-stimulated cell cycle progression following serum and cytokine
starvation. As expected, IL-6 stimulated cell cycle progression by both
cell lines after 24 and 48 h as revealed by the increased numbers
of cells with a DNA content suggestive of cells in S + G2/M
phases of the cell cycle (Fig. 1,
A and B; Table I).
Addition of IFN-
to the ANBL-6 cells failed to stimulate cell cycle
progression at any of the time points beyond that observed in control
cultures. In striking contrast, KAS-6/1 cells progressed into both the
S and the G2/M phases of the cell cycle in response to
IFN-
with increasing lengths of stimulation (Fig. 1B).
Moreover, IFN-
did not inhibit IL-6-stimulated KAS-6/1 cell entry
into either S or G2/M phases. These data suggest that
IFN-
-mediated growth control of myeloma cells is coupled to
regulation of cell cycle progression.
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Effects of IFN- on the Phosphorylation Status of pRb--
We
next examined the possibility that the differential effects of IFN-
on the growth of these cell lines resulted from differential phosphorylation of pRb. As shown in Fig.
2A, IL-6 induced the hyperphosphorylation of pRb in both cell lines as revealed by the
reduced rate of pRb electrophoretic migration. When the effects of
IFN-
on the ANBL-6 cell line were examined, it was first noted that
a 12-h stimulation with IFN-
failed to inactivate pRb by hyperphosphorylation in this cell line and second that IFN-
profoundly inhibited IL-6-stimulated hyperphosphorylation of pRb. By
contrast, the hyperphosphorylated form of pRb predominated in
IFN-
-stimulated KAS-6/1 cells. The level of pRb hyperphosphorylation
stimulated by the combined addition of IL-6 and IFN-
was comparable
with that observed in cells stimulated with IL-6 alone (results not shown).
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IFN- Modulates the Expression of Cyclin D in Myeloma Cell
Lines--
It is likely that alterations in the expression of cyclin D
or CDK2 and CDK4 might contribute to either the induction or inhibition of pRb phosphorylation by IFN-
. To test this possibility, we next
analyzed expression of cyclin D and CDKs in cells cultured with IL-6 or
IFN-
for 6 h. As may be seen in Fig. 2B, IL-6
stimulated the increased expression of cyclin D in both cell lines.
Densitometric scanning of multiple experiments revealed that IL-6
increased cyclin D expression by 6.0 ± 0.8-fold in the ANBL-6
cells (n = 2) and by 9.4 ± 0.5-fold in the
KAS-6/1 cells (n = 3). By contrast, IFN-
treatment
of the ANBL-6 cell line resulted in markedly reduced levels of cyclin D
expression (0.2 ± 0.1 relative to control cell levels,
n = 2). Furthermore, the IL-6-induced increase in
cyclin D expression was antagonized by coculturing ANBL-6 cells with IFN-
(0.6 ± 0.1 relative to control cell levels,
n = 2). IFN-
stimulation of the KAS-6/1 cells,
however, resulted in an increase in cyclin D expression (6.7 ± 1.3-fold induction over unstimulated cells, n = 3).
When the blot was reprobed for the expression of CDK4, no apparent
modulation in expression was observed in control or IFN-
-stimulated
cells (Fig. 2C). Similar results were obtained when CDK2
levels were quantitated (results not shown).
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Kinetics of CDK4 and CDK2 Activity Correlates with G1
to S Phase Progression--
pRb phosphorylation during early and late
G1 phase is coordinated by the activation of CDK4 and CDK2,
respectively (17). To confirm the role of CDK4 and CDK2 in the time
course of pRb phosphorylation, we next assayed kinase activity in CDK4
and CDK2 immunoprecipitates from ANBL-6 and KAS-6/1 cell lysates (Fig. 4). At 8 h poststimulation, IL-6
stimulated CDK4 activity in ANBL-6 cells as indicated by a 4.4 ± 0.1-fold (n = 2) increase in phosphorylation of a
GST-Rb fusion protein. Although IFN- treatment of the ANBL-6 cells
did not alter CDK4 activity over that observed in unstimulated cells
(0.7 ± 0.2 relative to control cell levels, n = 2), it suppressed IL-6-induced CDK4 activation to the level of activity
observed in unstimulated cells. In contrast, CDK4 kinase activity was
increased 4.1 ± 0.2-fold over control cells (n = 2) in response to IFN-
as well as IL-6 (7.0 ± 1.6-fold
increase over control cells, n = 2) in KAS-6/1 cells.
When CDK2 activity was assayed in the ANBL-6 cells (Fig.
5), a 24-h IL-6 stimulation increased
CDK2 activity by 8-9-fold over that of unstimulated cells. Although
IFN-
itself did not affect basal CDK2 activity, it inhibited the
IL-6-mediated increase in CDK2 activity. In contrast, both IL-6 and
IFN-
induced a significant increase in CDK2 activity in the KAS-6/1
cell line when assayed after 24 h. Unlike the rapid alterations in
the expression of cyclin D, modulations in CDK2 activity were observed
only after the effects on cell cycle progression and pRb
phosphorylation became obvious.
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Role of CDK Inhibitors in IFN- Responsiveness--
Because
IFN-
has been shown to induce p21 expression (29), we next asked
whether IFN-
stimulation could also affect p21 expression in myeloma
cells. As shown in Fig. 6, neither IL-6 nor IFN-
significantly affected p21 expression levels in either the
ANBL-6 cells or KAS-6/1 cells. Similarly, p27kip1 expression was not
modulated by IL-6 or IFN-
in either cell line (data not shown).
Thus, expression levels of p21Cip1 or p27kip1 do not correlate with pRb
phosphorylation status.
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DISCUSSION |
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The mechanisms that underlie the variable response of patient
myeloma cells to IFN- treatment have remained elusive primarily as a
result of a lack of a suitable model system that is representative of
in vivo heterogeneity. Our panel of myeloma cell lines,
however, circumvents this difficulty and provides a physiologically
relevant model system in which to study the inherent heterogeneity in
IFN-
responsiveness. Moreover, the KAS-6/1 cell line, which uniquely proliferates in response to IFN-
, provides us with a tool to understand "nonclassical" IFN-
responses in a human cell line. Our previous results showed that despite the contrasting effects of
IFN-
on the growth of the myeloma cell lines ANBL-6 and KAS-6/1, no
differential activation of STAT factors was observed nor was any
autocrine IL-6 expression induced in the cells that were growth stimulated by IFN-
(24). These results prompted us to examine other
mechanisms that may account for the differential biological responsiveness to IFN-
. The studies described herein demonstrate that one of the key mechanisms underlying this heterogeneity may involve regulation of genes that encode for cell cycle regulatory molecules. Our results show that IFN-
stimulates the differential induction of cyclin D2 and p19 INK4d.
Our analysis of cell cycle regulatory molecules was prompted by a
series of observations. We have demonstrated that IFN--stimulated proliferation was accompanied by hyperphosphorylation of pRb and that
this did not occur in the growth-inhibited cell line, ANBL-6. Moreover,
IFN-
was capable of antagonizing IL-6-stimulated pRb phosphorylation
in the same cell line. The finding that IFN-
inhibits
IL-6-stimulated pRb phosphorylation in the ANBL-6 cells and directly
stimulates pRb hyperphosphorylation in the KAS-6/1 cells is
interesting, as these events precede the appearance of alterations in
cell cycle progression and thymidine incorporation. This suggested that
modulation of the phosphorylation status of pRb may be a key event in
determining whether IFN-
stimulates growth or is growth inhibitory.
Our results are consistent with other reports in which sensitivity to
growth inhibition by IFN-
correlated with suppression of pRb
hyperphosphorylation (19, 20). However, the KAS-6/1 cells differ from
cells resistant to the inhibitory effects of IFN-
in that these
cells are growth responsive to IFN-
. IFN-
stimulates pRb
inactivation, which may allow KAS-6/1 cells to pass this critical
G1 to S phase checkpoint.
One striking observation in our study was the ability of IFN- to
up-regulate cyclin D2 protein expression in the growth-responsive KAS-6/1 cells but not in the growth-inhibited ANBL-6 cells. In results
not shown, we have also demonstrated that IFN-
up-regulated cyclin
D2 mRNA in the KAS-6/1 cells but not in the ANBL-6 cells. The
cyclin D2 promoter contains potential binding sites for transcription factors of the AP1 family, CTF/NFY, MyB, and C/EBP (30). Therefore, it
is possible that IFN-
activates one or more of these transcription factors in the KAS-6/1 cells via a mitogenic signaling pathway. We are
currently investigating this possibility. We have also shown that
IFN-
stimulates an increase in CDK4 and CDK2 activity, which
correlated with G1 to S phase progression of the KAS-6/1 cells. Because the activation of CDKs requires association with cyclins, cyclin D2 may therefore play an important role in the regulation of G1 progression by activating CDK4.
Another means of cell cycle regulation is provided by the Cip1/Kip1 and
INK4 family CKIs. p21Cip1 has been shown to be involved in
IFN--mediated growth arrest via STAT1-dependent
transcription of p21 waf1/cip1 (29). This suggested the possibility
that the differential effects of IFN-
on myeloma cell proliferation
may result from differential regulation of p21. However, we did not observe any difference in the expression of p21 with IFN-
stimulation. As we have previously observed STAT1 activation in both
cell lines following IFN-
stimulation (24), our observations are
consistent with the notion that p21 transcription following STAT
activation may not differ between the ANBL-6 and KAS-6/1 cell lines.
Our results also do not support a role for p27kip1 in initiating growth arrest in response to IFN.
Our analysis of INK4 family expression levels did not show any
alterations in p15, p16, and p18 mRNA and protein expression in
myeloma cells in response to IFN-. Although p18 has been shown to be
a potential CKI in terminal differentiation of myoblasts (31) or
EBV-transformed B cells (32), it does not seem to function as a primary
CKI in the growth arrest of undifferentiated cycling cells. Our data
support these observations as myeloma cells differ from normal plasma
cells because their terminal differentiation is deregulated.
Of interest, p19 expression was induced at both RNA and protein levels
by IFN- in the ANBL-6 cells by as early as 2 h poststimulation, whereas in the IFN-
growth-responsive KAS-6/1 cells, p19 levels remained unaltered following stimulation by IFN-
. These data suggest
that IFN-
may regulate growth of myeloma cell lines by inducing
transcription of p19 as an early response gene prior to the
G1 arrest. As IFN-
also induced p19 expression in the presence of IL-6 in the ANBL-6 cells (data not shown), it suggests that
one of the mechanisms by which IFN-
may inhibit
IL-6-dependent proliferation is by inducing p19 expression.
Recent work by Chan et al. (33) demonstrated the ability of
p19 to interact with the Nur77 orphan steroid receptor using a yeast
two-hybrid system. Although an in vivo interaction between
Nur77 and p19 has not been formally demonstrated, these data raise the
possibility that p19 might directly bind to targets other than cyclin
D-dependent kinases. It is conceivable, therefore, that the
basis for p19 specificity resides within its ability to simultaneously
contact secondary targets such as transcription factors. Thus, p19
induction in response to IFN-
may lead to functional modulation of a
transcription factor besides pRb. The mechanisms underlying the
induction of p19 expression in the ANBL-6 cells by IFN-
, as well as
the mechanisms underlying the failure of IFN-
to induce p19 in the
KAS-6/1 cells, are currently under investigation.
In summary, we have demonstrated that IFN--stimulated myeloma cell
proliferation or growth arrest correlates with induction of cyclin D2
or p19, or lack thereof. Because of the important role that both of
these proteins play in up- or down-regulation of the cell cycle, these
results are highly suggestive that these molecules play a direct role
in determining cellular outcome in response to IFN-
. Although these
results are very interesting, it is important to acknowledge that our
studies have not yet identified the signals that lie upstream of these
effects, which presumably lie in close proximity to the IFN-
receptor. In this regard, a serine/threonine kinase PKR has been shown
to down-regulate c-Myc expression in growth-arrested cells (34).
However, we have not observed alterations in the expression of c-Myc in
growth inhibitory or proliferative responses of myeloma cells to
IFN-
(35). It will be interesting to determine whether PKR or
additional signaling pathways may control induction of p19 or cyclin
D2. Finally, it remains possible that genetic variations in myeloma cell lines may lead to IFN-
-dependent activation of
different transcription factors, which ultimately results in positive
or negative regulation of p19 and cyclin D2 and hence in differential growth control.
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ACKNOWLEDGEMENTS |
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We thank Drs. Robert T. Abraham and Gregory J. Brunn for the generous gift of CDK2 antisera and helpful discussions. We also acknowledge the technical expertise of Renee C. Tschumper and Bonnie K. Arendt.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant CA-62228 from the National Cancer Institute.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 Immunology,
Mayo Clinic/Foundation, 200 1st St. S.W., Rochester, MN 55905. Tel.:
507-284-5617; Fax: 507-266-0981; E-mail:
jelinek.diane{at}mayo.edu.
1
The abbreviations used are: IFN-,
interferon-
; CDK, cyclin-dependent kinase; CKI, CDK
inhibitor; MM, multiple myeloma; PAGE, polyacrylamide gel
electrophoresis; PCR, polymerase chain reaction; pRb, retinoblastoma
protein; STAT, signal transducer and activator of transcription; IL-6,
interleukin-6; mAb, monoclonal antibodies; DTT, dithiothreitol; bp,
base pair(s); GST, glutathione S-transferase.
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REFERENCES |
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