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John D. Dingell Veterans Affairs Medical Center, Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201
Abstract
Signal transduction via modulation of phosphorylation after
selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears
to play a role in okadaic acid (OA)-mediated effects. Treatment of
several estrogen receptor-negative human breast carcinoma (HBC) cells
with 100 nM OA resulted in induction of
c-fos, c-myc, and cyclin-dependent kinase
inhibitor p21WAF1/CIP1 genes. Transfections
of various luciferase reporter constructs in HBC cells revealed
involvement of activator protein-1-dependent as well as -independent
pathways in induction of the c-fos gene by OA.
MDA-MB-468 HBC cells were stably transfected with plasmids expressing
luciferase, chimeric luciferase- c-fos 3'
untranslated region (3'UTR), or chimeric
luciferase-p21WAF1/CIP1 3'UTR mRNAs.
Expression of chimeric luciferase-c-fos and
luciferase-p21WAF1/CIP1 mRNAs was elevated
by OA in several independent sublines. Actinomycin D chase experiments
revealed an enhanced rate of decay of luciferase-c-fos
mRNA, whereas treatment with OA caused 
3.5-fold enhanced stability
of the chimeric luciferase-c-fos mRNA only. By
transfecting different plasmids containing deletions of
c-fos 3'UTR, OA-responsive sequences were mapped to an
86-nucleotide, AU-rich region. UV cross-linking experiments using HBC
cell cytosolic proteins showed multiple complexes with the AU-rich
region subfragments of c-fos, as well as
c-myc and p21WAF1/CIP1
mRNAs. OA enhanced binding of a novel Mr
75,000 protein present in the cytosolic extracts of HBC cells
to the AU-rich RNA probes of all of the above three genes. Taken
together, OA regulation of HBC cell gene expression involves the
activator protein-1 pathway, as well as enhanced binding of a novel
Mr 75,000 protein to an AU-rich region of
the 3'UTRs of the target genes.
Introduction
OA3 is a polyether compound of a C38 fatty acid that belongs to a new category of tumor-promoting agents that do not activate protein kinase C, yet can cause an increase in the phosphorylation of cellular proteins because of its inhibition of PP1 and PP2A (1) . Tumor-promoting effects of OA are presumably attributable to increased phosphorylation of one or more proteins that serve as substrates for protein kinase C and are dephosphorylated by PP1 and/or PP2A. Numerous studies have shown the usefulness of OA in understanding novel cellular processes that are regulated by phosphorylation (2) .
OA regulates gene expression at transcriptional,
posttranscriptional, or posttranslational levels. For example, exposure
of human skin fibroblasts to OA resulted in transcriptional repression
of elastin (3)
and type I and type III collagen genes
(4)
, whereas several genes including collagenase,
stromelysin (Ref. 4
and references within),
c-Jun, Egr, and IL-6 (Ref. 5
and
references within) were transcriptionally induced in human skin
fibroblasts in the presence of OA. IL-8 gene induction in
the presence of OA involves both transcriptional (via a nuclear
factor-
B site in the promoter of the IL-8 gene) as well
as posttranscriptional (enhancement of stability of the IL-8
message) mechanisms in the HL-60 cells (6)
. Similarly,
OA-dependent enhanced expression of nerve growth factor gene
in the rat cortical astrocytes has also been shown to be at both the
transcriptional as well as posttranscriptional levels (7)
,
whereas OA treatment of mouse P19 embryonal carcinoma cells leads to
reduced stability of neurofilament L gene mRNA
(8)
. In NIH3T3 cells, OA has been shown to cause increased
phosphorylation of p53 (9)
, whereas OA-mediated inhibition
of phosphorylation of retinoblastoma gene product was noted in the
mouse embryonic fibroblasts (10)
, and OA also caused
repression of expression and kinase activities of the cell cycle
regulatory proteins cdc2 and cyclin A (11)
. OA-regulated
cyclin- dependent kinase inhibitor
p21WAF1/CIP1 gene at both the
transcriptional (via the AP-2 site in its promoter) as well as
posttranscriptional levels in K549 cells (Ref. 12
and
references within). Transcription of several other genes, including
heat shock protein 70 and manganese superoxide
dismutase, was induced by OA in N-18 mouse neuroblastoma cells and
HeLa cells, respectively (13
, 14)
. OA also caused
induction of the cellular proto-oncogene c-fos in different
cell types including MCF-7 HBC cells, where it has been shown to
involve transcriptional as well as posttranscriptional mechanisms
(15
, 16)
.
The present study was undertaken to further understand the molecular
basis of OA-dependent gene regulation with reference to the ER-negative
HBC cells. Cyclin-dependent kinase inhibitor
p21WAF1/CIP1 and cellular proto-oncogenes
c-fos and c-myc expression was elevated in
OA-treated HBC cells. We found that OA used transcriptional mechanisms
involving the AP-1 pathway, as well as posttranscriptional mRNA
stabilization mechanisms. An AU-rich, OA-responsive, 86-nucleotide
subfragment of c-fos 3'UTR was mapped by deletion analyses.
UV cross-linking and gel mobility shift experiments using radiolabeled
ARE subfragment probes of c-fos, as well as
p21WAF1/CIP1 and c-myc 3'UTRs,
revealed the presence of multiple RNA-protein complexes. Binding of a
novel Mr 75,000 protein to ARE
subfragment probes was significantly elevated in cytosolic extracts
derived from OA-treated HBC cells. We propose that elevated binding of
the putative Mr 75,000 protein to
the AREs of 3'UTRs constitutes one of the key mechanism(s) involved in
posttranscriptional regulation of gene expression by OA in the HBC
cells.
Results
OA Exposure Caused Induction of Expression of Several Genes in
ER-negative Breast Carcinoma Cells.
MDA-MB-468 and MDA-MB-435 HBC cells were exposed to 10-, 20-,
40-, 60-, 80-, and 120-nM doses of OA for a period of
7 h. Total RNAs were prepared from both the floating and adherent
cells, and expression of c-fos, c-myc, and
p21WAF1/CIP1 genes was evaluated by
Northern blot hybridization. As shown in Fig. 1
, the 120-nM dose of OA caused elevated expression
of the c-fos, p21WAF1/CIP1, and
c-myc genes in the HBC cells.
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were transiently transfected in HBC cells. The plasmids containing
p21WAF1/CIP1 promoter (WWP-Luc; Ref.
12
), GADD45 promoter (GADD-Luc 6.3; Ref.
17
), and c-fos promoter (Fos-Luc1) were used in
the first instance, because all of the three genes were elevated in the
presence of OA (see Fig. 1
and data not shown). As shown in Fig. 3, A
and B, treatment with OA caused 2–3-fold
increase in luciferase activities in the case of plasmids containing
the GADD45 promoter and c-fos promoter. The
plasmid WWP-Luc- containing promoter for the
p21WAF1/CIP1 gene, however, showed 2-fold
enhancement of luciferase activity in the presence of OA only in
MDA-MB-435 HBC cells. Because AP-2 elements present in the promoter of
the p21WAF1/CIP1 gene have been
demonstrated previously to be involved in the OA-dependent induction of
p21WAF1/CIP1 in A549 cells
(12)
, it would appear that additional, perhaps novel,
mechanisms exist for OA-dependent induction of the
p21WAF1/CIP1 gene in HBC cells.
|
|
40 h after
transfection; both the controls and treated cultures were harvested at
48 h and assayed for luciferase activities. All of the constructs
elicited an 2.5–3-fold increase in the luciferase activities in the
presence of OA (Fig. 3C)
. In addition, Fos-SV40-Luc clone
4.12 and pGL2-Promoter plasmids were independently and transiently
transfected into MDA-MB-468 HBC cells, followed by treatment of cells
with 100 nM OA as described above. Fos-SV40-Luc
clone 4.12 did not elicit OA-dependent enhancement of luciferase
activities when compared with the pGL2-Promoter construct (data not
shown). CMV-Luc clone 6.1 (19)
,
(AP-1)5-CMV-Luc, CMV-Luc-Fos clone 5.2,
(AP-1)5-CMV-Luc-Fos, and CMV-Luc-Fos clone 5A.7
plasmids were independently and transiently transfected into HBC cells.
Approximately 40 h after transfection, the cells were treated with
100 nM OA for an additional 7 h; the
untreated and OA-treated cells were harvested, followed by assays for
luciferase activities. As shown in Fig. 3, D
and
G, CMV-promoter driven luciferase activities were induced
2-fold by OA. However, plasmids containing the AP-1 element
([AP-1]5-CMV-Luc) or c-fos 3'UTR
sequences (CMV-Luc-Fos 5.2) elicited additional 2–3-fold enhanced
luciferase activities in the presence of OA. Because the
c-fos promoter has been shown to contain AP-1 elements,
OA-dependent enhanced luciferase activities of the CMV-Luc-Fos 5.2
construct suggested the possibility of additional AP-1-independent
pathways. Furthermore, the plasmid containing AP-1 elements as well as
the entire 3'UTR of the c-fos mRNA
([AP-1]5-CMV-Luc-Fos) elicited a 5–6-fold
increase in luciferase activities in the presence of OA (Fig. 3D)
, suggesting that pathways using AP-1 elements and 3'UTR
sequences cooperate in OA-mediated induction of the c-fos
gene in HBC cells. In addition, as shown in Fig. 3
E, the
plasmid CMV-Luc-Fos 5A.7 showed reduced activities in the presence of
OA when compared with plasmid CMV-Luc-Fos 5.2, indicating that the
cis elements present in 0.8-kbp 3'UTR fragment
involved in OA-dependent up-regulation of the c-fos gene
were functional in their sense orientation. Thus, the OA-responsive
sequences of c-fos 3'UTR are unlikely to be transcriptional
in their function, because sequences mediating transcriptional
repression or enhancement of a given gene are known to function
independently of their orientation and location from the transcription
start site. The plasmid CMV-Luc clone 6.1 also elicited an 2-fold
increase in the luciferase activity in the presence of OA when compared
with untreated controls, suggesting that OA indeed used multiple
mechanisms for up-regulation of different genes in the HBC cells. The
OA-responsive cis sequences present in the c-fos
3'UTR were further mapped by using various deletion constructs (see
Fig. 2
). OA caused enhanced luciferase activities of all of the
constructs except CMV-Luc 6.1 and CMV-Luc-Fos 9.11 (Fig. 3F)
, suggesting that OA-responsive elements were located
within the 86-nucleotide ARE of the 3'UTR of the c-fos gene.
Thus, in addition to AP-1 transactivation, the data in Fig. 3
demonstrate that cis elements encompassed in the 3'UTR of
the c-fos gene were used in the OA-dependent regulation of
the c-fos gene in the ER-negative HBC cells.
OA Caused Posttranscriptional Regulation of c-fos
and p21WAF1/CIP1 Genes.
To further elucidate OA-mediated posttranscriptional regulatory
mechanism(s), MDA-MB-468 HBC cells were transfected with either CMV-Luc
6.1 (19)
, CMV-Luc-Fos 5.2, or CMV-Luc-WAF 4.2
(19)
, followed by generation of several independent stable
sublines in the presence of G418. Two independent sublines derived from
stable integration of each of the above plasmids, expressing low to
moderate levels of the respective chimeric RNAs, were treated with 100
nM OA for a period of 5 h. OA-treated as well as
untreated cells were harvested, followed by extraction of total
cellular RNAs and analysis of expression of luciferase mRNAs by
Northern blot hybridization. As shown in Fig. 4
A, expression of chimeric luciferase-c-fos
(Lanes 1 and 3) as well as luciferase-waf
(Lanes 9 and 11) mRNAs was reduced when compared
with the expression of luciferase mRNA (Lanes 5 and
7). Thus, the presence of the 3'UTRs of the c-fos
and p21WAF1/CIP1 genes down-regulated
expression of the CMV-Promoter-driven luciferase reporter gene in the
HBC cells. Furthermore, expression of chimeric
luciferase-c-fos (Lanes 2 and 4) and
luciferase-waf (Lanes 10 and 12) mRNAs was
elevated by 6–7-fold in the cells treated with OA (Fig. 4)
. Taken
together, the data in Fig. 4
suggested that 3'UTR subfragments of
c-fos and p21WAF1/CIP1, present
in plasmid clones CMV-Luc-Fos 5.2 and CMV-Luc-WAF 4.2, contained
OA-responsive cis-regulatory sequences.
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shows that the presence of OA enhanced the stability of the chimeric
luciferase-c-fos mRNA by 3.5-fold in the HBC cell
sublines derived from transfection of plasmid CMV-Luc-Fos 5.2
(t1/2 of 75 min for control
versus t1/2 of 270 min
for OA-treated cells).
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) in conjunction with OA-treated and untreated HBC
cell cytosolic protein extracts. Several HBC cell cytosolic proteins
interacted with c-fos ARE (Fig. 6A)
. These include four major complexes of approximately
Mr 37,000–40,000,
Mr 51,000,
Mr 75,000, and
Mr 118,000 sizes (Fig. 6
A,
Lanes 2 and 5). The presence of an 200-fold
excess of an unlabeled c-fos ARE RNA abolished binding of
the above cytosolic proteins to the c-fos probe (Fig. 6
A, Lanes 3 and 6). However, the presence of an
200-fold molar excess of a nonspecific, unlabeled RNA derived from
plasmid pBSK-GADD 12.4 (see Fig. 2
) failed to compete for any
RNA-protein binding (Fig. 6
A, Lanes 4 and 7),
suggesting that the binding of the above cytosolic proteins to the
radiolabeled ARE probe of c-fos 3'UTR was specific. The
RNA-protein interactions using cytosolic proteins derived from
OA-treated HBC cells showed elevated binding of the above-noted
Mr 38,000–40,000 complex as well as
the Mr 75,000 complex with the
c-fos ARE probe, whereas the binding of the putative
Mr 118,000 complex was reduced (Fig. 6
A, Lanes 5 and 7).
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B, pretreatment of extracts with proteinase K (Lanes
3 and 6) or alkaline phosphatase (Lanes 4
and 7) abrogated RNA-protein interactions, indicating that
different proteins binding with c-fos 3'UTR AREs were
phosphoproteins.
OA Caused Elevated Binding of a Novel Mr
75,000 HBC Cytosolic Protein to the AU-rich Sequences of Different
Target Genes.
The interactions of the cytosolic proteins derived from
OA-treated HBC cells with the probes containing AREs of additional OA
target genes, including c-myc and
p21WAF1/CIP1, was further evaluated. The
in vitro binding of the HBC cell cytosolic proteins to the
radiolabeled 160-nucleotide p21WAF1/CIP1
RNA containing AREs (derived from the plasmid pBSK-Waf7A.1; see Fig. 2
)
was studied as described in "Materials and Methods." Four major
complexes of the approximate sizes noted above in the case of the
c-fos probe (Fig. 6A)
also bound with the
p21WAF1/CIP1 probe (Fig. 7
A, Lanes 2, 4, 5, and 7). The binding of the above
cytosolic proteins to the radiolabeled
p21WAF1/CIP1 3'UTR probe was specific,
because the presence of an 200-fold excess of unlabeled
p21WAF1/CIP1 probe RNA abolished
RNA-protein complexes (Fig. 7
A, Lanes 3 and 6),
whereas an 200-fold excess of a nonspecific, unlabeled RNA (derived
from plasmid pBSK-GADD 12.4; see Fig. 2
) failed to compete for any of
the complexes (Fig. 7
A, Lanes 4 and 7).
Nevertheless, the RNA-protein interactions using cytosolic proteins
derived from OA-treated HBC cells showed significantly elevated binding
of the above-noted Mr 75,000 complex
with the p21WAF1/CIP1 probe, whereas the
binding of the putative Mr 118,000
complex was reduced (Fig. 7
A, Lanes 5 and 7).
Taken together, the data in Figs. 6A
and 7A
suggest that the OA-dependent elevated binding of the putative
Mr 75,000 complex, as well as reduced
binding of the putative Mr 118,000
complex to the ARE probes, was involved in the regulation of
c-fos and p21WAF1/CIP1 genes in
the HBC cells. However, the OA-dependent elevated binding of the
putative Mr 38,000–40,000 complexes
with the c-fos ARE RNA probe in Fig. 6A
indicated
a possibility of a sequence-specific RNA-protein interaction that may
be involved in the OA-dependent regulation of the c-fos gene
in HBC cells.
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shows the presence of the above-noted four major
RNA-protein complexes of approximately
Mr 37,000–40,000,
Mr 51,000,
Mr 75,000, and
Mr 118,000 sizes in the case of URE
(Lanes 6 and 7) and c-myc RNA
(Lanes 10 and 11) probes. Fig. 7B
also
shows that the presence of an 200-fold excess of unlabeled,
110-nucleotide-long c-fos ARE RNA abolished binding of the
above cytosolic proteins to either c-fos (Lane
4), URE (Lane 8), or c-myc (Lane
12) probes. The cytosolic proteins derived from OA-treated HBC
cells showed elevated binding of the above-noted
Mr 38,000–40,000 complex with all of
the above three probes (Fig. 6
B, Lanes 3, 7, and
11). However, as noted before in Figs. 6A
and 7
A, the binding of the Mr
75,000 and Mr 118,000 proteins present
in the OA-treated HBC cell extracts was modulated in the case of
c-fos and c-myc RNA probes but not when URE probe
was used (Fig. 7
B, compare Lanes 3 and
11 with Lane 7). The data in Fig. 7
, therefore,
strongly suggest that OA-dependent elevated RNA-protein interactions
involving the putative Mr 75,000
protein with the c-fos, p21WAF1/CIP1, and
c-myc AU-rich RNAs consisted of novel protein that was
different than the protein observed in the
Mr 75,000 complex noted with the
URE probe. Furthermore, the binding of the
Mr 118,000 complex was also noted with
all of the probes used in Fig. 7
. However, reduced binding of the
putative Mr 118,000 complex was noted
in the case of cytosolic extracts derived from OA-treated HBC cells
when either c-fos or c-myc (Fig. 7
B, Lanes
3 and 11) ARE probes were used but not when URE probe
was used (Fig. 7
B, Lane 7). Discussion
In the present investigation, we found that submicromolar concentrations of OA caused elevated expression of several mRNAs including p21WAF1/CIP1, c-myc, and c-fos in HBC cells. OA also caused induction of GADD45 and early- response genes c-jun, egr-1, and NAK1. The elevated expression of c-fos, p21WAF1/CIP1, and GADD45 by OA was also noted at the individual protein levels (data not shown). Both the ER-negative MDA-MB-231 and MDA-MB-468 HBC cells possess a mutant, nonfunctional p53 (25) . Thus, OA- dependent up-regulation of p53-responsive p21WAF1/CIP1 and GADD45 genes in various HBC cells used in this study further corroborates the earlier observation (16) that wild-type p53 function was not necessary for the mechanism(s) of OA-dependent gene regulation.
The upstream promoter of the c-fos gene has been shown to
contain several regulatory sequences including SRE, sis-inducible
element, Ets motif adjoining SRE, and AP-1/ATF sites in the proximity
of SRE (Ref. 26
and references within). The cellular
retinoblastoma susceptibility gene protein Rb also binds to specific
cis elements in the c-fos gene promoter, leading
to repression of c-fos transcription (27)
. We
found that OA induction of c-fos gene was mediated, in part,
by the elements present in the c-fos promoter, and given the
similar levels of enhancement of (AP-1)5-CMV-Luc
activities by OA (see Fig. 3
), it was likely that OA activation of the
c-fos promoter involved AP-1 cis-trans
interactions.
In this report, we present the evidence, for the first time,
implicating the role of 3'UTR in induction of the c-fos gene
in the HBC cells. We demonstrate that OA caused significant
posttranscriptional stabilization of chimeric luciferase mRNA
containing entire 3'UTR sequences of the c-fos gene.
Deletion mapping revealed that the ARE of the c-fos 3'UTR
mediated, in part, OA-dependent induction of the c-fos gene
in HBC cells (see Fig. 3
). AREs (5'-AUUUA-3') located in the 3'UTR have
been shown previously to regulate expression of several genes including
c-fos, c-myc, and
p21WAF1/CIP1 at posttranscriptional levels
(Refs. 23
and 28
and references within).
Indeed, the presence of c-fos 3'UTR caused reduced levels of
the chimeric luciferase-c-fos as well as chimeric
luciferase-p21WAF1/CIP1 mRNAs, when
compared with the luciferase mRNA expressed in the different HBC
sublines with stably integrated plasmids CMV-Luc-Fos 5.2, CMV-Luc-WAF
4.2, or CMV-Luc 6.1.
Recently, ARE binding proteins from several labile mRNAs and
different cell types have been characterized. For example, a
Mr 32,000 protein present in both
nuclear and cytosolic extracts of HeLa cells has been shown to bind ARE
of different labile mRNAs including c-fos (20)
.
Another Mr 37,000–40,000 size ARE
binding protein (AUF-1) has also been shown to be present in both the
nuclear and cytosolic extracts of K562 cells (29)
. In
addition, heterogeneous nuclear ribonucleoproteins A1 and C of
Mr 36,000 and
Mr 43,000 size, respectively, and the
Mr 30,000 inducible factor called
AU-B, have been shown to bind AREs and were found to be cytoplasmic in
nature (30
, 31)
. Furthermore, several Elav-like proteins
have been demonstrated to bind AU-rich elements, and a
Mr 36,000 protein named HuR binds AREs
of c-fos mRNA and can be induced to redistribute from
nucleus to cytosol (21
, 23)
. Indeed, several of the
nuclear proteins derived from OA-treated HBC cells belonging to a size
range of Mr 35,000–60,000 showed
elevated binding to the c-fos ARE probe obtained from
plasmid pBSK-Fos 10.1 (data not shown). Whether the ARE binding HBC
cell nuclear proteins ranging from Mr
35,000–60,000 size consist of novel and/or the previously identified
RNA binding nuclear proteins, including heterogeneous nuclear
ribonucleoproteins (31)
, AU-B (30)
, and
Elav-like proteins (23)
, along with their precise role in
OA-dependent mRNA stabilization in HBC cells, remains to be determined.
Nevertheless, similar to the data shown in Figs. 6
and 7
,
Mr 75,000 protein complex also
demonstrated elevated binding to the c-fos ARE probe when
nuclear extracts derived from OA-treated HBC cells were used (not
shown). Thus, the underlying conservation and differences observed in
this study among the HBC cell cytosolic and nuclear proteins
interacting with c-fos ARE probe may point to the existence
of multiple levels of gene regulation by OA. Nevertheless, the HBC cell
ARE binding proteins were found to be phosphoproteins. Because OA
treatment has been shown previously to enhance phosphorylation of
cellular proteins (15)
, it is possible that OA-dependent
modulation of phosphorylation of the HBC cell ARE binding proteins may
constitute, in part, the basis for induction of c-fos,
c-myc, and p21WAF1/CIP1 genes.
The requirement for ARE in the OA-dependent stabilization of
c-fos was highlighted when a 20-nucleotide URE sequence of
c-fos 3'UTR failed to show OA-dependent elevated binding of
the putative Mr 75,000 HBC cell
cytosolic protein. It should, however, be noted that the above
20-nucleotide URE has been shown previously to bind, in
vitro, with three complexes C1, C2, and C3 having RNA-binding
proteins of Mr 37,000,
Mr 65,000–71,000, and
Mr 85,000 sizes, respectively
(21)
. We did not find elevated binding of the
Mr 65,000–71,000 or
Mr 85,000 complexes to the similar
20-nucleotide URE probe when cytosolic extracts obtained from
OA-treated HBC cells were used (see Fig. 7B
). Therefore, the
OA-dependent induction of different genes in HBC cells may use
interactions of novel and/or known proteins with the OA-responsive
3'UTR sequences of the target genes in a fashion independent of the
involvement of URE.
In conclusion, we report the existence of multiple mechanisms for
OA-dependent regulation of the early-response gene c-fos,
the oncogene c-myc, and the cell cycle-related genes
p21WAF1/CIP1 and GADD45 in the
ER-negative HBC cells. The evidence, including the data in this report,
suggests that transcriptional mechanisms underlying OA-mediated
regulation of HBC cell gene expression involve, in general,
AP-1-dependent pathways. OA also uses additional posttranscriptional
mechanisms of gene regulation by modulating half-lives of the target
mRNAs. The posttranscriptional control mechanisms involve specific
RNA-protein cis-trans interactions including
elevated binding of a novel Mr
75,000 cytosolic protein to the AREs in the 3'UTRs of the target
genes.
Materials and Methods
Materials.
DMEM, Ham’s F-12 medium, and fetal bovine serum were purchased from
Life Technologies, Inc. (Grand Island, NY). The oligonucleotides for
PCR amplification (see below) were purchased from either Bio-Synthesis,
Inc. (Lewisville, TX) or integrated DNA Technologies, Inc. (Coralville,
IA). AmpliTaq DNA polymerase and deoxynucleotides were obtained from
Perkin-Elmer/Cetus (Norwalk, CT). The restriction endonucleases and the
DNA modification enzymes were purchased from either Bethesda Research
Laboratories (Bethesda, MD) or New England Biolabs (Beverley, MA). The
reagents for dideoxy sequencing were purchased from United States
Biochemical Corp. (Cleveland, OH). OA was purchased from Alexis
Biochemicals (San Diego, CA).
Plasmids and cDNA Probes.
The plasmids containing full-length human
p21WAF1/CIP1 cDNA and human
p21WAF1/CIP1 promoter luciferase reporter
clone WWP-Luc (12)
were kindly provided by Drs. Ken
Kinzler and Bert Vogelstein (Johns Hopkins University, Baltimore, MD).
Fos-cat plasmid containing -356 to +109 of the human c-fos
promoter (27)
was obtained from Dr. Paul Robbins
(University of Pittsburgh, Pittsburgh, PA), and the plasmid pBR322
containing the entire human c-fos gene (32)
was
obtained from Dr. T. Curran (St. Jude’s Children Research Hospital,
Memphis, TN), whereas (AP-1)5-SV40-CAT plasmid
(33)
was provided by Dr. D. Schadendorf (Humboldt
University, Berlin, Germany).
Cell Lines and Cell Culture.
The ER-negative MDA-MB-468 HBC cells were provided by Dr. Anne
Hamburger (Greenebaum Cancer Center, University of Maryland, Baltimore,
MD). The ER-negative MDA-MB-435 HBC cell line was obtained from Dr.
Steven Byers (Lombardi Cancer Center, Washington, DC). The cells were
cultured routinely in DMEM/Ham’s F-12 medium (1:1) supplemented with
5% fetal bovine serum as described previously (19)
. Cells
were transiently transfected with various plasmids; ß-galactosidase
and luciferase activities were measured essentially as described
previously (19)
. In certain cases, appropriate doses of OA
were added 6–7 h prior to harvesting.
RNA Isolation and Northern Blot Analysis.
Logarithmically growing HBC cells were treated with different doses of
OA for 7 h. Expression of different genes in untreated as well as
OA-treated cells was studied by Northern blot hybridization essentially
as described before (19
, 34)
.
Cloning of Plasmid Constructs.
The BglII fragment containing five tandem repeats of the
AP-1 cis element was excised from the plasmid
(AP-1)5-SV40-CAT (33)
and ligated
into the unique BglII site upstream of the CMV promoter in
the CMV-Luc plasmid clone 6.1 (19)
to obtain plasmid
(AP-1)5-CMV-Luc as per the methods described
before (34)
. Cloning of the plasmid expressing the CMV
promoter-driven chimeric luciferase-p21WAF1/CIP1
3'UTR mRNA (CMV-Luc 4.2) was described previously (19)
.
The human GADD45 gene promoter luciferase reporter construct
GADD-Luc 6.3 has been described before (17)
.
Various c-fos Promoter-luciferase reporter clones were
obtained as follows. The c-fos promoter (0.5 kbp) and the
exon 1 region from position -356 to +109 present in Fos-CAT plasmid
(32)
were subcloned into pGL-2Basic vector plasmid
(Promega) to obtain the c-fos promoter-luciferase reporter
clone Fos-Luc 1. Approximately 840 bp containing the human
c-fos promoter and exon 1 region (from positions -600 to
+240; Ref. 32
) were PCR amplified using plasmid
pc-fos(human)-1 (35)
as template and oligos fos-1
(5'-ACGAAGCTTCAGGTGGGCGCTGTGAAGCA-3', antisense oligo, positions
225–244; Ref. 32
) and fos-2 (5'-GCAGGAACAGTGCTAGTATT-3',
sense oligo, positions -600 to -580; Ref. 32
). It should
be noted, however, that the position +240 in the above 840-bp
c-fos promoter and exon 1 fragment corresponds to position
+109 in the exon 1 of the previously described clone Fos-CAT
(27)
. A three-way ligation was set up using
BglII (end-filled) and HindIII-cut pGL2-Basic
vector, EcoRI (end-filled) and XhoI-cut 1.3-kbp
5'UTR fragment derived from plasmid pc-fos (human)-1, and
HindIII-XhoI-cut 840-bp PCR-amplified fragment of
the c-fos gene to obtain the c-fos
promoter-luciferase reporter clone Fos-Luc 2.5. Fos-Luc 2.5 thus
contains the c-fos promoter from positions +109 to -2000
(32)
. Additional c-fos promoter-luciferase
reporter clone Fos-Luc3.1 was obtained by removing an 1.4-kbp,
XhoI fragment of clone Fos-Luc 2.5. The c-fos
promoter in the clone Fos-Luc 3.1, therefore, contains fos
promoter from positions +109 to -600 (32)
. Approximately
2.3 kbp of NotI plus NcoI subfragment of the
c-fos gene (+180 to +2531; Ref. 32
) was derived
from plasmid pc-fos (human)-1 (35)
, end-filled and cloned
in the antisense orientation upstream of the SV40 promoter in the
XhoI-cut, end-filled vector plasmid pGL2-Promoter (Promega)
to obtain Fos-SV40-Luc clone 4.12.
Different plasmids expressing chimeric luciferase-c-fos
mRNAs were generated as follows. A modified pCDNA3-luc vector clone 1
was derived from CMV-Luc plasmid 6.1 (19)
, following
standard manipulations to remove a 260-bp XbaI to
PvuII subfragment containing Sp-6 promoter and bovine growth
hormone signal sequences. 0.8 kbp of the 3'UTR immediately following
the translation termination codon in exon 4 of the c-fos
gene was PCR amplified using plasmid pc-fos(human)-1 (35)
as template and oligos fos-3 (5'-CGACTCGAGGCAGGGAAGGGGAGGCA-3', sense
oligo, positions 2733 to 2750; Ref. 32
) and fos-4
(5'-CGACTCGAGAGCACAAGGTTGGTCGC-3', antisense oligo, positions 3532 to
3549; Ref. 32
). It should be noted that oligo fos-4 lies
downstream of the polyadenylation signal sequences of the
c-fos gene. The XhoI-cut, PCR amplified, 3'UTR
fragment of the c-fos gene was cloned in the sense
orientation in the unique XhoI site located at the 3'end of
the luciferase gene in the modified pcDNA3-Luc vector clone 1 above to
obtain the CMV-Luc-Fos clone 5.2. In addition, the 0.8-kbp,
XhoI fragment of the c-fos 3'UTR was excised from
clone CMV-Luc-Fos clone 5.2 and cloned either in the antisense
orientation at the unique XhoI site downstream of the
luciferase reporter gene of CMV-Luc clone 6.1 to obtain CMV-Luc-Fos
clone 5A.7 or in the sense orientation at the unique XhoI
site of the above-described plasmid
(AP-1)5-CMV-Luc to obtain the
(AP-1)5-CMV-Luc-Fos plasmid.
Deletions of the 3'UTR of c-fos cDNA were generated by
PCR using oligo fos-4 in combination with either fos-6
(5'-CGACTCGAGGCAAAACGCATGGAGTGT-3', sense oligo, positions 2937 to
2954; Ref. 32
), fos-7 (5'-CGACTCGAGAACTACTGTGTTCCTGGC-3',
sense oligo, positions 3143 to 3160; Ref. 32
), fos-8
(5'-CGACTCGAGTTTCCATGAAAACGTTTT-3', sense oligo, positions 3343 to
3360; Ref. 32
), or fos-9
(5'-CGACTCGAGCCTTGAGGTCTTTTGACATG-3', sense oligo, positions 3429 to
3448; Ref. 32
). The resultant PCR fragments were cloned in
sense orientations at the unique XhoI site of the
above-described modified pcDNA3-Luc vector to obtain CMV-Luc-Fos clones
6.4, 7.1, 8.3, and 9.11. The 105-bp AU-rich subfragment of
c-fos 3'UTR (positions 3343–3448; Ref. 32
) was
PCR amplified using a combination of oligos fos-8 and fos-10. Oligo
fos-10 is antisense of oligo fos-9, except that the 5' end of oligo
fos-10 has an overhang for the XbaI site. The PCR-amplified,
XhoI + XbaI-digested, 105-bp AU-rich subfragment
was then cloned in plasmid vector pBSKII (Stratagene) to obtain
pBSK-Fos clone 10.1. The cDNA fragment of CMV-Luc-Fos clone 8.3 was
sequenced to confirm the validity of the c-fos insert. A
20-nucleotide-long URE (positions 3406 to 3425; Ref. 32
)
present in the c-fos 3'UTR was subcloned as a double
stranded fragment in vector plasmid pBSK as described below. Sense and
the corresponding antisense oligos, having 5' overhangs of 4
nucleotides for XhoI and XbaI sites,
respectively, were synthesized. The oligos were phosphorylated,
annealed, and ligated into XhoI and XbaI-cut
vector plasmid pBSK to obtain the pBSK-URE plasmid. The 160-bp, AU-rich
subfragment of the p21WAF1/CIP1 3'UTR
present in plasmid CMV-Luc-WAF clone 7.1 (19)
was further
subcloned in plasmid vector pBSKII (Stratagene) to obtain pBSK-Waf
clone 7A.1. The 45-bp subfragment of GADD45 5'UTR, present
in clone 11.1 (17)
, was subcloned in the pBSKII vector
plasmid to obtain the pBSK-GADD clone 12.4. All of the plasmids
described above are also schematically drawn in Fig. 2
.
Protein encoding an open reading frame of human c-myc
cDNA (36)
was reverse transcription-PCR amplified using
total RNA extracted from MDA-MB-468 HBC cells and a combination of
sense (5'-GCGGAATTCGACGATGCCCCTCAACGTT-3'; positions -5 to +15; Ref.
36
) and antisense oligos
(5'-GCGGAATTCCTTACGCACAAGAGTTCCG-3'; positions +1303 to +1321; Ref.
36
). The EcoRI-digested PCR product was cloned
in the sense orientation into the vector plasmid pCDNA3 (InVitrogen) to
obtain the pCDNA3-Myc plasmid. The identity of the c-myc
cDNA insert in plasmid pCDNA3-Myc was ascertained by partial sequencing
using T7 and Sp-6 primers. The c-myc
cDNA insert of plasmid pCDNA-Myc was subsequently used in Northern blot
hybridization analysis. The plasmid pMycSD3 (24)
containing 400 nucleotides of the c-myc 3'UTR, followed
by 100 nucleotides of poly(A) sequences, was kindly provided by Dr.
Gary Brewer (University of Medicine and Dentistry of New Jersey,
Piscataway, NJ).
Stable Transfections and Analysis of mRNA Decay.
MDA-MB-468 cells were transfected independently with CMV-Luc
clone 6.1, CMV-Luc-Fos 5.2, or CMV-Luc-WAF 4.2. Several
neomycin-resistant (800–1000 µg/ml) sublines from each of the
transfections were obtained as described before (17)
. The
expression of luciferase, chimeric luciferase-c-fos, or
chimeric luciferase-p21WAF1/CIP1
transcripts was evaluated by Northern blot hybridization using a
radiolabeled 0.6-kbp, HindIII-EcoRI luciferase
cDNA fragment derived from pGL2-Basic (Promega). Two or more
independent sublines containing stably integrated plasmid CMV-Luc 6.1
(19)
or CMV-Luc-Fos 5.2, which showed low to moderate
levels of luciferase expression, were selected for analysis of mRNA
decay. OA-treated and untreated clonal derivatives of MDA-MB-468 cells
were cultured in the presence of transcriptional inhibitor AMD (4
µg/ml) for various times to study the rates of decay of transfected
luciferase and chimeric luciferase-c-fos transcripts,
essentially per the methodology described before (17)
.
In Vitro Transcription and Electrophoretic Gel
Mobility Shift Assay.
Plasmids pBSK-Fos 10.1, pBSK-Waf 7A.1, and pBSK-GADD 12.4 were
separately linearized with XbaI, and
T7 promoter primer was used to synthesize
unlabeled sense strand RNAs as described before (37)
.
Plasmid pMycSD3 (24)
was linearized with
HindIII, and Sp-6 promoter primer was used to synthesize
unlabeled sense strand c-myc 3'UTR RNA. Plasmid pBSK-URE was
linearized with EagI, and T7 promoter
primer was used to synthesize URE RNA. The
[
-32P]UTP (3000 Ci/mmol; 10 mCi/ml;
Amersham) labeled RNAs (107 cpm/µg) of
c-myc 3'UTR, p21WAF1/CIP1 3'UTR,
20-nucleotide c-fos URE fragment, and the 20-nucleotide URE
containing the 110-nucleotide-long c-fos ARE were derived
independently from plasmids pMycSD3, pBSK-Waf7A.1, pBSK-URE, and
pBSK-Fos 10.1, respectively.
Cytoplasmic protein extracts from MDA-MB-468 HBC cells were
prepared essentially as described before (38)
. Twenty µg
of cytoplasmic protein/reaction were preincubated at room temperature
for 10 min with 200 ng/µl Escherichia coli tRNA and 1x
binding buffer containing 12 mM HEPES (pH 7.5), 5
mM MgCl2, 1.25
mM EDTA, 1.25 mM DTT, 155
mM KCl, and 10% glycerol in a total reaction
volume of 9 µl. In some cases, Proteinase K (Sigma; 5 µg/reaction),
calf intestinal alkaline phosphatase (BMB; 40 units/reaction), or 1
µl of 200 ng/µl of unlabeled sense strand RNAs was included in
the preincubation step. The specific competitor RNAs consisted of
either the 105-nucleotide, ARE RNA derived from plasmid pBSK-Fos 10.1
or the 159-nucleotide, ARE RNA derived from plasmid pBSK-Waf 7A.1. The
nonspecific competitor RNA consisted of 45 nucleotides of 5'UTR
subfragment of GADD45 mRNA derived from plasmid pBSK-GADD
12.4. Radiolabeled probe RNA (1 ng/reaction; 10,000 cpm) was
then added to the reaction mix and incubated at room temperature for an
additional 30 min. The reactions were then exposed to UV on ice for 5
min (120 microjoules/min) using a UV cross-linker (Stratagene) to
cross-link the proteins interacting with the RNAs. Five units of RNase
T1 (BMB) and 2.5 units of RNase A (Sigma) were added to each reaction
and incubated at room temperature for 30 min. Fifty µg of Heparin
(Sigma) were added to each reaction, followed by incubation on ice for
10 min. The reactions were boiled and then analyzed on 12%
SDS-polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8) at 65 V in
1x Tris glycine buffer for a period of 14–16 h. The gels were dried
and exposed for autoradiography for appropriate period of times.
Acknowledgments
We are grateful to Jordan Denner and Richard Milanich of the Baltimore Veterans Affairs Medical center, Baltimore, MD, and William Browning of the John D. Dingell Veterans Affairs Medical Center, Detroit, MI, for preparing the illustrations.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Part of this work was carried out at
Greenebaum Cancer Center, University of Maryland, Baltimore, MD, and
was supported by the University of Maryland Institutional research
grant (to A. K. R.). Additional support was provided by Detroit
Medical Center Institute for Oncology and Allied Diseases institutional
research grant (to A. K. R.), the medical research services of the
Department of Veterans Affairs (to J. A. F.), and NIH Grant CA63335
(to J. A. F.). 
2 To whom requests for reprints should be
addressed, at John D. Dingell Veterans Affairs Medical Center, Research
151, Room B 4270, 4646 John R. Street, Detroit, MI 48201. Phone:
(313) 576-1000, extension 4492; Fax: (313) 576-1112; E-mail: Rishia{at}Karmanos.org
3 The abbreviations used are: OA, okadaic acid;
PP, protein phosphatase; AMD, actinomycin D; HBC, human breast
carcinoma cell; ER, estrogen receptor; UTR, untranslated region; ARE,
AU-rich region; URE, U-rich region; IL, interleukin; AP-1, activator
protein-1; CMV, cytomegalovirus; SRE, serum response element; oligo,
oligonucleotide.
Received for publication 8/ 1/00. Accepted for publication 9/ 5/00.
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