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Tumor Biology |
in Prostate Cancer Cells1
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on the neu/HER-2
proto-oncogene in the DU145, LNCaP, and PC-3 prostate cancer cell
lines. IFN- inhibited cell proliferation in both DU145 and PC-3
cells in a dose-dependent manner, whereas no inhibition of
proliferation was seen in LNCaP cells. Correspondingly, IFN-
treatment of DU145 and PC-3 cells resulted in an increased production
of the cyclin-dependent kinase inhibitor p21WAF1, whereas
no increase in p21WAF1 was seen in LNCaP cells. In
addition, IFN- induced phosphorylation of signal transducer and
activator of transcription (STAT) 1 in DU145 and PC-3 cells, but not in
LNCaP cells. Consistent with these findings, we found that IFN-
treatment of DU145 and PC-3 cells caused a reduction in neu/HER-2
expression, with no change seen in the LNCaP cell line. Transfection
and overexpression of the transcriptional coactivator p300 in PC-3
cells suppressed the reduction in neu/HER-2 expression after IFN-
treatment, suggesting a role for p300 in neu/HER-2 expression. The
antiproliferative activity and p21WAF1 production of these
cells after IFN- treatment were found to be reduced as well. We
propose that the down-regulation of neu/HER-2 by IFN- occurs via the
interaction of phosphorylated STAT1 with p300 because IFN-
activities requiring phosphorylated STAT1 are reduced in cells
overexpressing p300. These findings suggest that neu/HER-2 may play a
role in the growth of some prostate cancers and that IFN- may
suppress such cancers by down-regulation of neu/HER-2. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibition of cellular
proliferation occurs via the induction of the
CKI3
p21WAF1 (1, 2)
.
Like other cytokines, IFNs use the JAK/STAT signal transduction
pathway. IFN- binding of its cell surface receptor specifically
induces STAT1 phosphorylation, resulting in translocation to the
nucleus, where it binds specific -activated sites in target genes
(3)
. Recent studies have shown that both dimerized
phosphorylated STAT1 and unphosphorylated STAT1 monomer can bind the
CBP p300 (4)
. The CBP p300 family of proteins is a group
of transcriptional coactivators that can interact with a variety of
DNA-binding factors (5)
. p300 was originally isolated and
cloned because of its interaction with the E1A protein of adenovirus
(6)
. E1A does not directly bind DNA but regulates gene
expression via interactions with multiple transcription factors
(7)
. As a result, E1A is responsible for reducing p300
coactivation of several genes controlling activities such as cellular
differentiation, cell cycle progression, and transformation
(8)
.
The neu/HER-2 proto-oncogene has been linked to several human
malignancies including breast, ovarian, and prostate cancer
(9–12)
. Recently, it has been shown that neu/HER-2
promoter activity can be repressed by E1A (13)
.
Furthermore, this repression was reversed both by increasing amounts of
p300 as well as by normal amounts of p300 defective in E1A binding
(14)
. These findings suggest that p300 may play a role in
the regulation of the neu/HER-2 gene. Interestingly,
phosphorylated STAT1 homodimers from IFN--treated extracts have also
been shown to bind the E1A binding site of p300, indicating the
potential for IFN- to repress p300-regulated genes via
phosphorylated STAT1 (4)
. The experiments described in
this report are directed at IFN--induced down-regulation of the
neu-HER-2 receptor and the possible role of phosphorylated STAT1 in
this down-regulation via interaction with p300, analogous to E1A
down-regulation of neu/HER-2.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(specific activity, 1–4 x 107 units/ml) was obtained from Genzyme
Diagnostics (Cambridge, MA). The DU145, LNCaP, and PC-3 cell lines were
obtained from American Type Culture Collection (Manassas, VA). Complete
media for the DU145, LNCaP, and PC-3 cell lines consisted of Eagle’s
MEM, Eagle’s MEM + 1 mM sodium pyruvate
and 2 mM L-glutamate, and F12K medium,
respectively, supplemented with 10% fetal bovine serum, 200 units/ml
penicillin, and 200 µg of streptomycin. Antibodies to
p21WAF1, STAT1, and p300 were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Antibodies to phosphorylated STAT1
were obtained from New England BioLabs, Inc. (Beverly, MA). Antibodies
to neu/HER-2 were obtained from Upstate Biotechnology (Lake Placid,
NY). The p300 plasmid pRc/RSV-p300 containing a neomycin resistance
gene allowing for selection of colonies in Geneticin was generously
provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health
Sciences University, Portland, OR). The pRc/RSV plasmid was purchased
from Invitrogen (Carlsbad, CA). Transfectam reagent was obtained from
Promega (Madison, WI).
Transfection.
PC-3 cells were transfected with the plasmids pRc/RSV and pRc/RSV-p300
after the Transfectam reagent transfection protocol. PC-3 cells
(1.3 x 105) were plated in a
100-mm2 plate. A mixture of either pRc/RSV or
pRc/RSV-p300 DNA and Transfectam reagent was added to the PC-3 cells
and left in contact with the cells for 2 h. Transfected PC-3 cells
were then selected via incubation with complete media + 600
µg/ml Geneticin (Sigma Chemical Co., St. Louis, MO).
Antiproliferation Assay.
IFN- inhibition of cell number was determined by plating DU145,
LNCaP, PC-3, and PC-3(pRc/RSV-p300) cells (4–10 x 104 cells/well) into 6-well plates with or
without 500–20,000 units/ml IFN-. At 72 h, cells were
trypsinized, washed, and counted. Cell counts were performed using a
hemocytometer, and cell viability was assessed by trypan blue dye
(0.4%) exclusion.
Immunoprecipitation and Immunoblotting.
For preparation of cell lysates, DU145, LNCaP, PC-3, and
PC-3(pRc/RSV-p300) cells (4–6 x 106 cells/sample) were grown in the presence or
absence of 500–20,000 units/ml IFN- for various lengths of time and
lysed at 4°C for 20 min as described previously (1)
.
Equal amounts of protein from cell lysates were either used directly
for Western transfer (100–200 µg/sample) or subsequently
immunoprecipitated (375–500 µg/500 µl) with 1 µg of an antibody
specific for the protein of interest. After Western transfer, membranes
were immunoblotted with antibodies specific for the protein of interest
and developed using enhanced chemiluminescence (Amersham, Arlington
Heights, IL). Densitometric analysis of radiographic film using IA-200
Image Analysis Software was used to determine the percentage decrease
or fold-increase between band intensities based on total pixel values.
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RESULTS |
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on the
DU145, LNCaP, and PC-3 prostate cancer cell lines by determining the
total number of cells grown in the presence or absence of 0, 500, 1000,
and 5000 units/ml IFN- for 72 h. IFN- inhibited DU145 and
PC-3 cell growth by up to 59.1% and 50.8%, respectively, whereas no
inhibition of LNCaP cells was seen (Table 1
). We also found that IFN- inhibited DU145 and PC-3 cells in a
dose-dependent manner. Thus, the proliferation of the DU145 and PC-3
cell lines is inhibited by IFN- treatment, whereas LNCaP cells do
not appear to be affected.
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was due to up-regulation of the CKI
p21WAF1 (2)
.
p21WAF1 regulates the mammalian cell cycle by
directly binding cyclin-dependent kinases, which are necessary for cell
cycle progression, and blocking their kinase activity
(15)
. IFN--induced up-regulation of
p21WAF1 describes a mechanism by which IFNs
inhibit the cell cycle and subsequent cell growth. Asynchronous DU145,
LNCaP, and PC-3 cells were treated with 5000 units/ml IFN- for 16
and 24 h and subsequently lysed and immunoblotted. IFN- induced
p21WAF1 production in both DU145 and PC-3 cells,
but not in LNCaP cells (Fig. 1
). Consistent with our previous reports, IFN- up-regulation of
p21WAF1 correlates with inhibition of cell
proliferation.
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was intact in the DU145, LNCaP, and PC-3 cell lines. IFNs initiate
their cellular effects by binding to specific cell surface receptors.
Like other cytokines, this ligand-receptor interaction induces the
phosphorylation of a group of proteins known as STATs, which, in the
absence of ligand, reside unphosphorylated in the cytoplasm
(3)
. IFN--mediated signal transduction specifically
phosphorylates STAT1, which then acts directly to turn on
IFN--inducible genes. DU145, LNCaP, and PC-3 cells were treated with
5000 units/ml IFN- for 10 min. Cell lysates were immunoprecipitated
using antibodies specific for STAT1, and precipitates were subsequently
immunoblotted with antibodies for both STAT1 and phosphorylated STAT1.
Both DU145 and PC-3 cells had strong phosphorylation of STAT1 with
IFN- treatment, whereas no STAT1 phosphorylation was seen in LNCaP
cells (Fig. 2
). These results explain the lack of response by the LNCaP cell line to
IFN- because the JAK/STAT transduction pathway is not triggered in
these cells. However, the transduction pathway is active in both DU145
and PC-3 cells, consistent with up-regulation of
p21WAF1 and the associated antiproliferative
effects seen in these cells.
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on down-regulation of neu/HER-2
expression in the DU145, LNCaP, and PC-3 cell lines as a possible
mechanism for inhibition of cell proliferation. Lysates from cells
treated in the presence or absence of 5000 units/ml IFN- for 96 h were immunoblotted using antibodies specific for neu/HER-2. In DU145
and PC-3 cells, we saw a 61.6% and 78.7% reduction, respectively, in
neu/HER-2 expression, whereas no reduction in neu/HER-2 was seen in
LNCaP cells (Fig. 3A
). In addition, we saw a dose-dependent decrease in
neu/HER-2 expression in DU145 and PC-3 cells (Fig. 3B
).
Cells were treated with 500, 1000, and 5000 units/ml IFN- for
96 h, and lysates were immunoblotted. Thus, IFN- reduces
neu/HER-2 expression in both the DU145 and PC-3 cells, whereas no
reduction is seen in the LNCaP cell line, consistent with the failure
to phosphorylate STAT1 and induce p21WAF1 in the
latter cell line.
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-treated lysates binds p300 at the
E1A binding site (4)
. We therefore wanted to determine
whether p300 was involved in IFN--induced down-regulation of
neu/HER-2 expression. PC-3 cells were transfected with a plasmid
containing the p300 gene (pRc/RSV-p300). Transfected and wild-type PC-3
cells were then grown in the presence or absence of 5000 units/ml
IFN- for 96 h. Lysates were subsequently immunoblotted using
antibodies specific for both p300 and neu/HER-2. Transfected cells
showed a 30.7% increase in p300 (determined by densitometric analysis)
as compared with wild-type cells (Fig. 4A
). Furthermore, this increase in p300 production appeared to
affect neu/HER-2 expression after IFN- treatment because transfected
cells showed only a 27.7% reduction in neu/HER-2 expression as
compared with a 78.7% reduction in wild-type cells (Fig. 4B
). This suggests that p300 may play a role in
IFN--induced down-regulation of neu/HER-2.
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-induced down-regulation of neu/HER-2 after IFN- treatment,
we wanted to determine whether the overexpression of p300 interfered
with the antiproliferative effects of IFN-. Transfected PC-3 cells
were treated with or without IFN- at concentrations of 5,000 and
20,000 units/ml for a period of 72 h. Cells were then harvested,
and antiproliferative activity was determined by direct cell count.
Transfected cell growth was much less inhibited after treatment with
5,000 units/ml IFN- as compared with wild-type PC-3 cells, with
inhibition values of 7.1% versus 50.8%, respectively
(Table 2
). Interestingly, treatment of transfected cells with 20,000 units/ml
IFN- inhibited cell growth by 44.6%, which is comparable to maximum
inhibition levels seen in wild-type PC-3 cells. Thus, the
antiproliferative effect of IFN- is significantly reduced in PC-3
cells overexpressing p300 but is restored at higher concentrations of
IFN-. This suggests a dynamic and reversible interaction between
IFN--induced phosphorylated STAT1 and p300.
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. Because IFN- has reduced antiproliferative activity on PC-3
cells overexpressing p300, we determined the ability of IFN- to
up-regulate p21WAF1 in transfected PC-3 cells.
p300-transfected PC-3 cells were treated with or without IFN- at
concentrations of 5,000 and 20,000 units/ml over a time period of 16
and 24 h. Cells treated for 24 h with 5,000 and 20,000
units/ml IFN- showed a 1.5- and 3.7-fold increase in
p21WAF1 production, respectively, as determined
by densitometric analysis (Fig. 5
). No p21WAF1 was detected at 16 h. Wild-type
PC-3 cells treated with 5,000 units/ml IFN- over a 24-h period
showed a 2.5-fold increase in p21WAF1 production.
Thus, treatment of transfected PC-3 cells with 5,000 units/ml IFN-
led to decreased p21WAF1 production as compared
with wild-type PC-3 cells, correlating with the reduced
antiproliferative effects of IFN- on transfected PC-3 cells.
Consistent with the antiproliferative effects of IFN- on transfected
PC-3 cells, treatment with an increased concentration of IFN-
(20,000 unit/ml) was able to restore
p21WAF1 levels to those seen after IFN-
treatment of wild-type PC-3 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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exerts its effects through the JAK/STAT signal transduction pathway, in
which STAT1 molecules are phosphorylated and translocated to the
nucleus, where they bind to -activated sites and activate
transcription of target genes. Here we have shown that IFN- exhibits
strong antiproliferative effects on the prostate cancer cell lines
DU145 and PC-3. Correspondingly, treatment of these cell lines with
IFN- resulted in an increased production of the CKI
p21WAF1 as compared with media control.
Interestingly, IFN- treatment of the prostate cancer cell line LNCaP
did not result in any growth inhibition or increase in
p21WAF1 production. Examination of the JAK/STAT
signal transduction pathway showed equal amounts of constitutive STAT1
produced in all cell lines after IFN- treatment. However,
IFN--treated LNCaP cells failed to produce any phosphorylated STAT1,
whereas significant amounts were produced in both DU145 and PC-3 cells.
Independent studies have shown that phosphorylated STAT1 molecules
specifically recognize STAT-responsive elements in the
p21WAF1 promoter and are required for cell growth
inhibition in response to IFN- in various cell lines
(18)
. This correlates with the lack of antiproliferative
activity and failure to induce p21WAF1 in
LNCaP cells after IFN- treatment.
In addition to increasing p21WAF1 production, we
have found that IFN- treatment of DU145 and PC-3 cells results in a
dose-dependent down-regulation of the neu/HER-2 proto-oncogene,
although this was not seen in LNCaP cells. Recent studies have
suggested that the transcriptional coactivator p300 may play a role in
the regulation of neu/HER-2 (13)
. The adenovirus E1A
protein was found to transcriptionally repress the neu/HER-2 promoter.
Additional studies have shown that overexpression of p300 was able to
overcome the repression of neu/HER-2 promoter activity by E1A
(14)
. This repression was also overcome by a p300 mutant
deficient in E1A binding. These studies suggest that E1A binding to
p300 results in repression of neu/HER-2 promoter activity. In a
separate study, phosphorylated STAT1 from IFN--treated cell extracts
was found to bind to the E1A-binding domain of p300 (4)
.
In light of this evidence, we looked at the effect of p300
overexpression on IFN--induced down-regulation of neu/HER-2
expression in PC-3(pRc/RSV-p300) cells. Interestingly, the
overexpression of p300 was sufficient to significantly overcome the
repression of neu/HER-2 expression after IFN- treatment, as seen in
the case of neu/HER-2 repression by E1A. Thus, p300 appears to play a
role in the expression of neu/HER-2. Furthermore, IFN--mediated
repression of neu/HER-2 expression may occur via the interaction of
phosphorylated STAT1 with p300 because phosphorylated STAT1 has been
shown to bind to the E1A-binding domain of p300. This provides a
mechanism by which IFN- regulates neu/HER-2 expression and cell
growth via phosphorylated STAT1 in addition to
p21WAF1 inhibition of cyclin-dependent kinases.
We subsequently found that the overexpression of p300 greatly reduced
the antiproliferative activity of IFN- on PC-3 cells, correlating
with the derepression of neu/HER-2. However, this antiproliferative
activity was restored after treatment with an increased concentration
of IFN-, thereby increasing the amount of phosphorylated STAT1
available for transcription of target genes. The overexpression of p300
in PC-3 cells also significantly inhibited the ability of IFN- to
increase p21WAF1 production. Consistent with
antiproliferation studies, IFN--induced increases in
p21WAF1 production were restored to levels seen
in wild-type PC-3 cells after treatment with an increased concentration
of IFN-. Therefore, increased concentrations of IFN- (and thus
increased levels of phosphorylated STAT1) reversed the repressed
IFN--induced activities requiring phosphorylated STAT1 in cells
overexpressing p300. These studies correlate with the proposal
of p300-phosphorylated STAT1 binding in response to IFN- and
inhibiting the function of both factors. All studies involving
PC-3(pRc/RSV-p300) cells were repeated in PC-3 cells transfected with
the empty vector, pRc/RSV. The results of these studies showed that
PC-3(pRc/RSV) cells behaved in a manner similar to that of wild-type
cells (data not shown).
In summary, we have shown that IFN--induced inhibition of prostate
cancer cell line growth is accompanied by an increased production of
the CKI p21WAF1 and decreased expression of the
proto-oncogene neu/HER-2. Our findings also implicate the
transcriptional coactivator p300 in the positive regulation of
neu/HER-2 gene expression. Furthermore, we propose that IFN--induced
down-regulation of neu/HER-2 may involve the binding of
phosphorylated STAT1 to p300, thereby inhibiting neu/HER-2 expression.
Future studies will be directed at elucidating the interactions between
this and other IFN-induced transcription factors and this coactivator.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA 38587 (to H. M. J.).
This article is Florida Experiment Station Journal Series Number
R-07610. S. L. K. and A. C. H. contributed equally to this
work. 
2 To whom requests for reprints should be
addressed, at Room 1052, Building 981, P. O. Box 110700, Department of
Microbiology and Cell Science, University of Florida, Gainesville, FL
32611. Phone: (352) 846-0968; Fax: (352) 392-5922; E-mail: johnsonh{at}ufl.edu
3 The abbreviations used are: IFN, interferon;
CKI, cyclin-dependent kinase inhibitor; STAT, signal transducer and
activator of transcription; JAK, Janus kinase; CBP, cAMP-responsive
element binding protein-binding protein.
Received 1/ 6/00. Accepted 5/12/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene, 17: 2973-2979, 1998.[Medline]
induction of p21WAF1 in prostate cancer cells: role in cell cycle, alteration of phenotype, and invasive potential. Int. J. Cancer, 77: 138-145, 1998.[Medline]
signaling. Proc. Natl. Acad. Sci. USA, 93: 15092-15096, 1996. through Stat1-dependent and -independent pathways. EMBO J, 19: 263-272, 2000.[Medline]
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