Identification of PRG1, A Novel Progestin-Responsive Gene with Sequence Homology to 6-Phosphofructo-2-Kinase/Fructose- 2,6-Bisphosphatase
Jenny A. Hamilton,
Michelle J. Callaghan,
Robert L. Sutherland and
Colin K. W. Watts
Cancer Research Program Garvan Institute of Medical
Research St. Vincents Hospital Sydney, New South Wales 2010,
Australia
 |
ABSTRACT
|
---|
To define early molecular targets of
progestin action, the differential display technique was used to
identify genes with altered levels of expression in T-47D breast cancer
cells treated with the synthetic progestin ORG 2058 for 3 h. PRG1
was first isolated as a 200-bp cDNA clone and its progestin regulation
confirmed by Northern analysis. Cloning of the complete coding region
of PRG1 revealed that it shared a high degree of amino acid sequence
identity with isoforms of the enzyme
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from several
tissues and species. Expression of PRG1 mRNA was observed in several
normal breast epithelial and breast cancer cell lines and in a variety
of human tissues, with highest expression in the breast, aorta, and
brain. In T-47D cells, PRG1 mRNA was rapidly and transiently induced by
progestins, expression peaking between 2 and 4 h and returning to
control levels by 12 h. Progestin-induced increases in PRG1 mRNA
were inhibited by the progestin antagonist RU 486 and occurred via the
progesterone receptor. Progestin induction of PRG1 mRNA was also
inhibited by actinomycin D but not by cycloheximide. PRG1 is therefore
a novel human gene that is directly regulated by progestins via the
progesterone receptor.
 |
INTRODUCTION
|
---|
The sex steroid hormone progesterone has two major roles in
mammalian physiology. First, progesterone is involved in preparing the
uterus for implantation of the fertilized ovum. Second, progestins have
proliferative and differentiating effects on mammary epithelium
(reviewed in Refs. 1 and 2). Mitotic activity in breast epithelium
varies in a cyclic manner through the menstrual cycle, and a role for
progesterone in this process is suggested by observations that levels
of this hormone and epithelial cell proliferation are both maximal
during the late secretory phase (3). Progesterone is essential for
lobuloalveolar development and preparation for lactation: when
ovulation is established, progesterone, produced by the corpus luteum,
stimulates growth of the lobuloalveolar structures and, during
pregnancy, promotes branching of the ductal system and differentiation
of alveolar cells into secretory cells ready for milk production. The
importance of progestin in these processes is clearly illustrated in
progesterone receptor (PR) knockout mice, which fail to develop
lobuloalveolar structures (4). Some breast tumors retain progesterone
responsiveness, and the use of high doses of synthetic progestins are
recognized endocrine therapies for PR-positive breast cancers, since in
this setting progestins have an antiproliferative effect (5).
Progestins also have predominantly growth-inhibitory effects on human
breast cancer cell lines in vitro, although under certain
conditions they may stimulate growth (Refs. 2 and 6 and references
therein). Mechanistic studies have clearly defined both a stimulatory
and inhibitory effect of progestins on breast cancer cell cycle
progression (6), but the functional consequences of these effects
in vivo remain to be defined. This is of considerable
importance given the widespread pharmacological usage of progestins in
oral contraceptives and in hormone replacement therapy.
The mechanisms underlying the biological effects of progestins in the
normal breast and in breast cancer are only partially understood.
Progestin action is mediated primarily via the PR, which upon
activation by ligand binding interacts with gene promoter sequences
containing progesterone responsive elements (PREs) to regulate gene
transcription. Very few mammalian genes have been described that are
directly regulated by progestins in this manner; examples include
c-jun (7), c-fos (8), fatty acid synthetase (9),
PR (10, 11), and uteroglobin (12, 13). Of these c-jun and
c-fos have roles in control of cell cycle progression. Other
progestin-regulated genes with known roles in cell cycle control are
c-myc (6, 8), and cyclin D1 (14). PR can be classified with
those progestin-regulated genes whose functions are related to steroid
and growth factor action and might contribute to the proliferative
effects of progestin, at least indirectly. This group includes estrogen
receptor (15), retinoic acid receptors (16), epidermal growth factor
receptor (6, 17), PRL receptor (18), insulin-like growth factor I
receptor (19), insulin receptor (20), epidermal growth factor (21),
transforming growth factors
and ß1 (6, 8, 22, 23),
17ß-hydroxysteroid dehydrogenase (24), and insulin-like growth
factor-binding proteins 4 and 5 (25). Other progestin-regulated genes
including fatty acid synthetase (9), alkaline phosphatase (26), and
lactate dehydrogenase (27) have functions that are important in
differentiation effects mediated by progestin. While progestin action
ultimately involves changes in the levels of large numbers of mRNAs and
proteins, many of these require intermediary de novo protein
synthesis. Specific genes that mediate the proliferative effects of
progestins are poorly defined, and thus much remains to be learned
about genes induced as an acute response to progestin treatment and
their role in mediating progestin effects on cell proliferation and
differentiation.
The aim of this study was therefore to identify novel
progestin-regulated target genes involved in early responses of
progestin action in breast cancer cells. We employed a serum-free cell
culture system using the T-47D human breast cancer cell line that
enables both stimulatory and inhibitory effects of progestins on cell
cycle progression to be observed (6). In this system progestins
accelerate entry into S phase of cells already progressing through
G1 phase, presumably by acting on genes or gene products
that are rate limiting for G1 progression. These cells
subsequently complete a round of replication and then become
growth-arrested early in G1 phase.
We isolated mRNA after 3 h of treatment with the synthetic
progestin ORG 2058 (16
-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione)
and used this as a template for cDNA synthesis and analysis by the
differential display technique (28). This time point was chosen as
previous studies showed that final commitment to cell cycle progression
does not occur until 3 h after initial progestin exposure (6, 14),
implying that critical changes in progestin-regulated gene expression
are most likely to be detected within this time frame. Using this
strategy we now report the identification and characterization of a
novel progestin-regulated cDNA from human breast cancer cells, PRG1
(progestin-responsive sequence, Garvan 1), which shares a high degree
of sequence homology with the bifunctional enzyme
phosphofructo-2-kinase/fructose-2,6-bisphosphatase1
and may link progestin action with the glycolytic pathway.
 |
RESULTS
|
---|
Cloning of a cDNA Identified by Differential Display
The differential display technique was used to identify mRNAs in
T-47D human breast cancer cells whose levels of expression had altered
in response to treatment with the synthetic progestin ORG 2058 for
3 h. Using the PCR primer combination 5'-T12GG and
5'-CAAACGTCGG, a total of nine cDNA fragments identified by gel
electrophoresis were clearly up-regulated (Fig. 1A
).
Preliminary confirmatory screening by Northern analysis showed one of
these, designated PIG1, was induced rapidly in the presence of ORG 2058
(Fig. 1B
) and therefore warranted further characterization.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Identification of Differentially Expressed cDNAs in
T-47D Cells Treated with the Synthetic Progestin ORG 2058
A, Identification of PIG1 by differential display. Total RNA obtained
from T-47D cells treated with ORG 2058 or vehicle control (ethanol) for
3 h was used as a template for differential display PCR reactions
with 5'-T12GG and 5'-CAAACGTCGG as primers. The PCR
products were separated on a 6% polyacrylamide denaturing gel, and the
gel was exposed to x-ray film. The arrows indicate PCR
products present at a higher level in the progestin-treated (T)
compared with control (C) lane. B, Confirmation of the progestin
induction of PIG1 by Northern blot analysis. T-47D cells proliferating
in insulin-supplemented serum-free medium were treated with 10
nM ORG 2058 (T) or ethanol vehicle (C) for 3 h, and
total RNA was harvested for Northern analysis. The Northern blot was
probed with the PIG1 fragment.
|
|
To obtain the complete coding sequence from which PIG1 was derived, a
human kidney cDNA library constructed using oligo-dT-primed and
random-primed cDNA was screened using the PIG1 fragment. Four cDNAs
were isolated after screening 2.85 x 105
recombinants, namely 11.2, 6.3, 3.1, and 19.1 (Fig. 2A
).
Further screening of this library with an oligonucleotide derived from
5'-sequence of 3.1 (5'-ACCGTCATCGTCATGGTGGG-3') and with a 373-bp
EcoRI-BglII restriction fragment derived from 3.1
resulted in the isolation of a chimeric clone 9.1 and clone 8.1 (Fig. 2A
). Screening of a human heart library with the 373-nt
EcoRI-BglII restriction fragment resulted in the
isolation of clone H7.1 (Fig. 2A
). Clones 11.2, 6.3, and 3.1 were
sequenced in their entirety on both strands, and 350 nucleotides of the
5'-end of clone 19.1 were sequenced on both strands, to give 2887
nucleotides of cDNA sequence, designated PRG1, shown in uppercase
letters (Fig. 2B
). While the 2887 nucleotides of sequence is less
than the mRNA size determined from Northern blots, it contains a
complete open reading frame (ORF) as discussed below.

View larger version (54K):
[in this window]
[in a new window]
|
Figure 2. Determination of the PRG1 cDNA Sequence
A, A schematic representation of PRG1 structure with a
restriction map for the PRG1 cDNA and the cDNA clones used to derive
the PRG1 sequence shown beneath. The initial PCR cDNA
fragment identified by differential display was designated PIG1. All
the cDNA clones were isolated from a human kidney cDNA library with the
exception of H7.1, which was isolated from a human heart cDNA library.
Clone 9.1 is a chimeric clone. The cosmid clone containing genomic
sequence, part of which overlaps with PRG1 cDNA sequence, was obtained
from the Genbank database and is shown above the PRG1
sequence. The numbers refer to distances in nucleotides.
B, Nucleotide and deduced amino acid sequence of PRG1. The nucleotide
sequence determined from cDNA clones is shown in uppercase
letters whereas the nucleotide sequence obtained from the
cosmid genomic clone CRI-JC2015 is shown in lowercase
letters. The translation termination codon is shown by an
asterisk in the amino acid sequence. The in-frame
termination codon that precedes the initiating methionine is
underlined. The numbers refer to
distances in nucleotides.
|
|
Comparison of the cDNA sequence with the GenBank and EMBL databases
revealed a partial overlap with a cosmid clone (CRI-JC2015) (29)
containing human genomic sequence from chromosome 10. Nucleotides
numbering 1399 of the cDNA overlap with nucleotides 16712070 from
the cosmid clone but in the reverse orientation (Fig. 2A
). The first
1670 nucleotides of the cosmid clone are not present in PRG1 and appear
to be intron sequence as the consensus splicing sequence (30) occurs
adjacent to PRG1 homologous sequence. Extrapolating backward 27
nucleotides from the 5'-end of the cDNA into the genomic sequence
reveals an in-frame stop codon (see Fig. 2B
) where the genomic sequence
is shown in lowercase letters.
Analysis of the PRG1 cDNA sequence identified an ORF containing 520
amino acids encoding a protein very similar to human liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2)
(31) as well as to bovine brain and heart forms of this enzyme (32, 33). PRG1 bears 72% amino acid identity with the human liver
PFK-2/FBPase-2 in 447-amino acid overlap and 93% and 74% identity
with bovine brain PFK-2/FBPase-2 and bovine heart PFK-2/FBPase-2 in
462-amino acid and 447-amino acid overlap, respectively (Fig. 3
). The ORF identified in the PRG1 cDNA sequence appears
to be complete. The initiation codon is preceded by an in-frame stop
codon located 357 nucleotides upstream as identified in the cosmid
clone (CRI-JC2015) and is surrounded by a consensus sequence for strong
translational initiation (34). In addition, the initiating methionine
is within close proximity to initiating methionines of other known
related proteins (e.g. Refs. 31, 33, 35, 36) with the
exception of the bovine brain PFK-2/FBPase-2, the cDNA sequence of
which has only been partially characterized (32).

View larger version (77K):
[in this window]
[in a new window]
|
Figure 3. Protein Sequence Homologies of Human and Bovine
PFK-2/FBPase-2 Isoforms
Amino acid sequence homology between PRG1 and PFK-2/FBPase-2 isoforms
from human liver, bovine brain, and bovine heart. An alignment of the
amino acid sequences was obtained using the computer programs MacVector
4.5.3 and SeqVu. Identical residues found in three or four of the
proteins are boxed. Amino acids are numbered from the
first residue. The bovine brain sequence is believed to be incomplete
(32). bov., Bovine; hum., human.
|
|
Analysis of the deduced amino acid sequence of PRG1 using the Prosite
Database Release 13.0 revealed several consensus phosphorylation sites
for cAMP/cGMP-dependent protein kinases, protein kinase C, casein
kinase II, and tyrosine kinases. Of particular interest was a consensus
cAMP-dependent protein kinase site at Ser461 and a consensus protein
kinase C site at Tyr471 because in the bovine heart isoform
cAMP-dependent phosphorylation at Ser466 and protein kinase C
phosphorylation at Tyr476 affects kinase activity (37). In addition,
the ATP/GTP-binding site signature motif, conserved in all mammalian
forms of PFK-2/FBPase-2 (38), was identified at amino acids 4249 as
was the phosphoglycerate mutase family phosphohistidine signature,
amino acids 5160.
Northern Blot Analysis of PRG1 Gene Expression
The expression profile of PRG1 in a panel of human breast cancer
and normal breast cell lines was investigated by hybridizing Northern
blots of total RNA isolated from one normal breast epithelial cell
strain (HMEC 184), two transformed epithelial cell lines (HMEC 184B5
and HBL-100), and 12 breast cancer cell lines (only nine are shown) to
a probe from a 1.8-kb cDNA subclone, 6.3 (Fig. 2A
), which contains 98%
of the PRG1 ORF. PRG1 mRNA, a single transcript of
4.4 kb, was
expressed in all the breast cancer and normal breast epithelial cell
lines examined (Fig. 4A
). The highest level of
expression was in the T-47D cell line, and the lowest levels were noted
in SK-BR-3 (not shown), BT-474, HBL-100, HMEC 184, and HMEC 184B5 cell
lines. There was no correlation between PRG1 mRNA expression and
estrogen receptor, PR, or glucocorticoid receptor (GR) status (39)
although the T-47D cell line, which expresses PR at a level 5-fold
higher than the other cell lines, had the highest level of expression
(40).

View larger version (42K):
[in this window]
[in a new window]
|
Figure 4. Expression of PRG1 mRNA in Different Human Tissues
and Breast Cancer and Normal Breast Cell Lines
A, Northern blot analysis of total RNA from different human breast
cancer and normal breast cell lines. The blot was probed with a 1.8-kb
cDNA subclone of PRG1 and an oligonucleotide complementary to 18S rRNA
as a loading control. B, Northern blot analysis of poly A+
RNA from human tissues. The blot was hybridized with a 1.8-kb cDNA
subclone of PRG1. Molecular sizes of markers are indicated. PBL,
Peripheral blood leukocytes. C, Dot blot analysis of poly
A+ RNA from human tissues. The blot was hybridized with a
1.8-kb cDNA subclone of PRG1. Row A: 1, whole brain; 2, amygdala; 3,
caudate nucleus; 4, cerebellum; 5, cerebral cortex; 6, frontal lobe; 7,
hippocampus; 8, medulla oblongata; Row B: 1, occipital lobe; 2,
putamen; 3, substantia nigra; 4, temporal lobe; 5, thalamus; 6,
subthalamic nucleus; 7, spinal cord; Row C: 1, heart; 2, aorta; 3,
skeletal muscle; 4, colon; 5, bladder; 6, uterus; 7, prostate; 8,
stomach; Row D; 1, testis; 2, ovary; 3, pancreas; 4, pituitary gland;
5, adrenal gland; 6, thyroid gland; 7, salivary gland; 8, mammary
gland; Row E: 1, kidney; 2, liver; 3, small intestine; 4, spleen; 5,
thymus; 6, peripheral leukocyte; 7, lymph node; 8, bone marrow; Row F:
1, appendix; 2, lung; 3, trachea; 4, placenta; Row G: 1, fetal brain;
2, fetal heart; 3, fetal kidney; 4, fetal liver; 5, fetal spleen; 6,
fetal thymus; 7, fetal lung.
|
|
The tissue specificity of PRG1 gene expression was investigated by
hybridizing Northern blots of poly A+ RNA isolated from a
variety of human tissues to a probe made from the subclone 6.3. A
4.4-kb transcript was detected in all the tissues examined (Fig. 4B
).
The apparent abundance of PRG1 mRNA in skeletal muscle shown in Fig. 4B
is likely the result of uneven mRNA loading because in a second set of
human tissue poly A+ Northern blots, skeletal muscle mRNA
levels were similar to those of the kidney. In the heart, a band of
1.35 kb was also detected. This band was more easily detected under
lower stringency conditions when it was also found to be present in
kidney, pancreas, skeletal muscle, and colon (data not shown) and
suggests that the cDNA PRG1 probe was cross-reacting with a related
sequence. A third transcript of around 9.5 kb was also detected in
skeletal muscle.
Hybridization of a human tissue poly A+ RNA dot blot
representing a wider range of tissues and loaded by the manufacturer so
as to allow a more accurate determination of relative mRNA abundance
(Fig. 4C
) showed that PRG1 expression was highest in mammary gland,
various brain tissues, and in the aorta. Moderate levels of expression
were seen in most other tissues, although several had relatively low
levels of expression, e.g. liver, colon, and bladder.
PRG1 mRNA Is Induced Transiently by Progestin
To examine in detail the kinetics of progestin induction of PRG1,
regulation of PRG1 mRNA expression was investigated in T-47D cells
cultured in insulin-supplemented serum-free medium and harvested for
mRNA at various time points after ORG 2058 treatment (Fig. 5
). PRG1 mRNA was detected in cells cultured in the
absence of ORG 2058. The induction of PRG1 mRNA by ORG 2058 was an
early and transient event. Maximal levels of PRG1 induction, 4.4-fold
relative to time-matched control in the experiment shown in Fig. 5
, were observed at 3 h following treatment. The induction at 3
h was typically between 3 and 4.4-fold relative to time-matched
controls. After 6 h mRNA levels had decreased and by 12 h had
returned almost to control levels. A more detailed analysis of early
time points showed that maximal levels were reached by 2 h and
sustained until 4 h (data not shown). The increase in PRG1
preceded increases in the proportion of cells in S phase (data not
shown), which typically occur around 10 h in this system (6).

View larger version (46K):
[in this window]
[in a new window]
|
Figure 5. Regulation of PRG1 mRNA Expression by the Synthetic
Progestin ORG 2058
T-47D cells proliferating in insulin-supplemented serum-free medium
were treated with 10 nM ORG 2058 (closed
circles) or ethanol vehicle (open circles), and
total RNA was harvested for Northern analysis. The Northern blot shown
in panel A was probed with a 1.8-kb cDNA subclone of PRG1 and with an
oligonucleotide complementary to 18S rRNA as a control for loading.
Positions of the 28S and 18S ribosomal bands are indicated. Values in
panel B were obtained by densitometric analysis of the autoradiograph
and are expressed relative to the control at 0 h.
|
|
Induction of PRG1 mRNA in Breast Cancer Cell Lines Is Mediated via
the PR
To determine whether the effects of ORG 2058 on PRG1 expression
were likely to be mediated by the PR, we examined the effects of other
synthetic progestins and the antiprogestin RU 486
(17ß-hydroxy-11ß-(4-methylaminophenyl)-17
-(1-propynyl)-estra-4,9-diene-3-one)
in a variety of breast cancer cell lines. T-47D cells, growing
exponentially in medium containing 5% FCS, were treated in parallel
with the synthetic progestins ORG 2058, R5020
(17
-21-dimethyl-19-norpregn-4,9-diene-3,20-dione), and MPA
(17
-acetoxy-6
-methyl-4-pregn-4-en-3,20-di-one)
at 10 nM and mRNA harvested at 3 h. All three
synthetic progestins induced PRG1 mRNA between 2- and 2.5-fold above
control levels (data not shown). PRG1 mRNA was also induced by the
synthetic progestin livial [ORG OD-14,
(7
,17
)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one, 10
nM] in T-47D cells growing in the presence of 5%
charcoal-treated FCS as discussed later. The effect of ORG 2058 on PRG1
mRNA in another PR-positive cell line (MCF-7) and in a PR-negative cell
line (MDA-MB-231) were investigated. ORG 2058 increased PRG1 mRNA in
MCF-7 cells approximately 2-fold above control mRNA levels at 3 h
(Fig. 6A
). In contrast, in MDA-MB-231 cells the levels
of PRG1 mRNA in the presence of ORG 2058 were decreased approximately
30% below control mRNA levels at 3 h, with recovery at 6 h
(Fig. 6B
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 6. Regulation of PRG1 mRNA Expression in MCF-7 and
MDA-MB-231 Cells by ORG 2058 and Dexamethasone
A, MCF-7 (PR +ve, GR +ve) and B, MDA-MB-231 (PR -ve, GR +ve) cells
proliferating in RPMI 1640 medium supplemented with 5% FCS were
treated with ORG 2058 (10 nM), dexamethasone (100
nM), or ethanol vehicle and harvested for Northern analysis
at 3 h and 6 h. Densitometric analysis of the Northern blot
hybridized with a 1.8-kb cDNA subclone of PRG1 is presented expressed
relative to control at 0 h and adjusted for loading. The data in
panel B were obtained from the mean of two experiments. C, MDA-MB-231
cells were transiently transfected with pMSG-CAT and treated with
dexamethasone (100 nM) ± RU 486 (100 nM), and
ORG 2058 (10 nM) ± RU 486 (100 nM), or ethanol
vehicle (control) for 48 h before harvesting and determination of
CAT activity. Results are expressed as fold-induction over control
levels of CAT activity and are the mean of triplicate determinations.
|
|
Figure 6
also demonstrates another important aspect of PRG1
regulation. Given that the consensus sequence for the glucocorticoid
and progesterone response elements is similar (41), one might
expect that glucocorticoids could regulate PRG1 expression via the GR.
The GR could also have a role in mediating progestin effects, given the
receptor cross-reactivity of some progestins for both PR and GR. The
MCF-7 and T-47D cell lines express GR, although at low levels (42), and
treatment with the synthetic glucocorticoid dexamethasone
(9-fluro-11,17,21-trihydroxy-16-methylpregn-1,4-diene-3,20-dione,
100 nM) for either 3 or 6 h produced no increase in
PRG1 mRNA (Fig. 6A
and data not shown). Using these lines we have
previously found that dexamethasone is also unable to induce either
expression of epidermal growth factor receptor (a
glucocorticoid-responsive gene in breast cancer cell lines) or
chloramphenicol acetyltransferase (CAT) activity from a transiently
transfected reporter construct, pMSG-CAT (which contains
glucocorticoid- and progestin-responsive sequences from the MMTV
promoter) (42, 43). These results, indicating an apparent absence of
functional GR in these cell lines, argue against a GR-mediated
mechanism for progestin regulation of PRG1. To establish whether the GR
can regulate PRG1 expression in a glucocorticoid-responsive cell line,
these experiments were repeated in MDA-MB-231 cells, which contain high
levels of GR but no PR (42). In these cells, dexamethasone reduced PRG1
mRNA to approximately 60% of control levels at 3 h with some
recovery evident at 6 h, suggesting a small glucocorticoid effect,
but opposite to that of progestins in PR-positive cell lines. GR
functionality in these cells was confirmed by transient transfection
with pMSG-CAT (Fig. 6C
), which showed 4- to 5-fold induction of CAT
activity after treatment with dexamethasone that could be inhibited by
the glucocorticoid/progestin antagonist RU 486. ORG 2058 (10
nM) did not induce CAT activity, indicating that this
progestin does not cross-react with the GR at this concentration and
that the small ORG 2058 effect on PRG1 expression in these cells is
unlikely to be mediated via the GR.
Additional evidence for the involvement of PR in mediating progestin
effects on PRG1 was obtained using the antagonist RU 486, which acts as
an antiprogestin by competitively inhibiting the binding of progestins
to the PR (44). Its effect on the induction of PRG1 mRNA was
investigated by treatment of T-47D cells with ORG 2058 (10
nM) and RU 486 (100 nM) either alone or
simultaneously in serum-free medium supplemented with insulin. The
cells were harvested 3 h after the treatment, when PRG1 mRNA
levels were at a maximum. Simultaneous administration of ORG 2058 and
RU 486 led to complete inhibition of progestin-induced PRG1 expression,
while treatment with RU 486 alone had no effect on mRNA levels (Fig. 7A
). Similar effects were seen in MCF-7 cells grown in
the presence of serum although the antagonist effect of RU 486 was not
quite as pronounced (Fig. 7B
). Together, these data are consistent with
the progestin effect being mediated via the PR and not the GR.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 7. Antagonism of ORG 2058 Induction of PRG1 mRNA by
the Antiprogestin RU 486
A, T-47D cells proliferating in insulin-supplemented serum-free medium
were treated with ORG 2058 (10 nM), RU 486 (100
nM), the two compounds simultaneously (ORG 2058 + RU 486),
or ethanol vehicle and harvested for Northern analysis at 3 h. The
Northern blot was probed with the PIG1 fragment of PRG1 and with an
oligonucleotide complementary to 18S rRNA as a control for loading. The
graph represents densitometric analysis of the
autoradiograph expressed relative to the control at 3 h. B, MCF-7
cells proliferating in medium supplemented with 5% FCS were treated
with ORG 2058 (10 nM), ORG 2058 + RU 486 (100
nM) simultaneously, or ethanol vehicle and harvested for
Northern analysis at 3 h. The Northern blot was probed with a
1.8-kb cDNA subclone of PRG1 and with an oligonucleotide complementary
to 18S rRNA as a control for loading.
|
|
Because progestin effects can potentially be mediated by the androgen
receptor (AR) present in breast cancer cell lines (39, 45), the effect
of the antiandrogen hydroxyflutamide
(
,
,
-trifluoro-2-methyl-4'-nitro-m-lactotoluidide, 1
µM) on ORG 2058 (10 nM) induction of PRG1 was
examined. No antagonism was observed (data not shown), an indication
that progestins do not mediate their effects on PRG1 induction via the
AR. In addition, 5
-dihydrotestosterone (1100 nM), with
or without a 100-fold molar excess of hydroxyflutamide, did not induce
PRG1 above basal levels, further evidence that this gene is not
AR-regulated in breast cancer cells (data not shown).
PRG1 Induction by Progestin Does Not Require de Novo
Protein Synthesis
To distinguish between direct activation of PRG1 transcription by
the PR or indirect activation via the synthesis of intermediary
proteins, T-47D cells were treated with ORG 2058 (10 nM) in
the presence of the protein synthesis inhibitor, cycloheximide (20
µg/ml). Cycloheximide failed to block the progestin-mediated
induction of PRG1 mRNA. Treatment with cycloheximide alone resulted in
an increase of PRG1 mRNA to a level similar to that achieved by ORG
2058 alone, while in the presence of both ORG 2058 and cycloheximide
there was superinduction of PRG1 mRNA (Fig. 8A
). The magnitude of this induction,
12-fold in the experiment shown in Fig. 8
, was larger than expected
from a combination of the responses to the individual compounds. Such
superinduction involving protein synthesis inhibitors is characteristic
of genes such as ß- and
-actin, c-fos, and
c-myc, which are induced early (02 h) after stimulation of
cells by mitogens (46, 47, 48). At time points up to 6 h, the transcription
inhibitor actinomycin D (5 µg/ml) prevented induction of PRG1 mRNA in
T-47D cells treated with the synthetic progestins livial (Fig. 8B
) or
ORG 2058 (Fig. 8C
). Together, these data suggest that the induction of
PRG1 mRNA is due to a direct effect of the PR on PRG1 transcription and
does not require de novo protein synthesis.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 8. Effect of Cycloheximide and Actinomycin D on
Progestin Induction of PRG1 mRNA
A, T-47D cells proliferating in insulin-supplemented serum-free medium
were treated with ORG 2058 (10 nM), cycloheximide (CHX, 20
µg/ml), ORG 2058, and CHX simultaneously or ethanol vehicle and
harvested for Northern analysis at 3 h. The Northern blot was
probed with the PIG1 fragment of PRG1 and with an oligonucleotide
complementary to 18S rRNA as a loading control. The
graph represents densitometric analysis of the
autoradiograph expressed relative to the control at 3 h. B, T-47D
cells proliferating in medium supple mented with 5% charcoal-treated
FCS were treated with
livial (10 nM) and ethanol
vehicle in the presence and absence of actinomycin D (5 µg/ml) and
harvested for Northern analysis. The Northern blot was probed with a
1.8-kb cDNA subclone of PRG1. C, Ethanol control; L, livial. C, T-47D
cells were treated as in panel B above, except that ORG 2058 was
substituted for livial. C, Ethanol control.
|
|
 |
DISCUSSION
|
---|
Despite the biological importance of progestins, the patterns of
gene expression mediating proliferative and other responses to these
steroids are not understood in detail. To define early molecular
targets of progestin action, the differential display technique was
used to identify genes with altered levels of expression in T-47D human
breast cancer cells treated with the synthetic progestin ORG 2058 for
3 h. A cDNA fragment, PIG1, cloned using this method, was used to
screen Northern blots and identified a 4.4-kb mRNA species that was
induced in response to a variety of synthetic progestins within 3
h. Compilation of DNA sequence data from several cDNA clones generated
a 2.8-kb cDNA, PRG1. The ORF of PRG1 contains 520 amino acids and
encodes a protein sharing a high degree of identity with
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2)
isoforms from many species, including that from human liver. The human
liver isoform of PFK-2/FBPase-2 (PFKFB1) has been assigned to
chromosome X (49) and on this basis, and because of significant
differences throughout the nucleotide and amino acid sequences, is
clearly distinct from PRG1, the sequence of which partially overlaps
with the cosmid clone, CRI-JC2015, derived from a genomic sequence from
chromosome 10 (29). A locus for an additional, uncharacterized human
gene that may correspond to the rat heart isoform (PFKFB2) has been
identified on chromosome 1 (49).
Six isoforms of PFK-2/FBPase-2 have been identified in mammalian
tissues. They are referred to as F (fetal)-, L (liver)-, M (muscle)-, H
(heart)-, T (testis)-, and B (brain)-type. Although multiple genes
exist in other species, e.g. the bovine H- and B-isoforms,
only one human gene, the L-isoform, has been cloned and functionally
characterized. The primary differences between mammalian isoforms of
this enzyme are the length of the amino- and carboxy-terminal regions
and the composition of these regions particularly with regard to the
number of protein phosphorylation sites (reviewed in Ref.38).
Phosphorylation reciprocally affects the enzymes kinase and
bisphosphatase activity. Although the ORF of PRG1 shares the highest
degree of identity with the bovine brain PFK-2/FBPase-2, its expression
is not restricted to neural tissue but is detectable in all tissues
examined, although at a wide range of levels. In addition, the 3'-end
of the PRG1 ORF sequence has greater similarity with bovine heart
PFK-2/FBPase-2 than with the bovine brain isoform. On the basis of the
above evidence, it is likely that PRG1 represents the third known human
gene of the PFK-2/FBPase-2 family. A comparison of their respective
tissue distributions suggests PRG1 has a much more widespread role than
the L-isoform of PFK-2/FBPase-2 whose expression among the tissues
shown in Fig. 4B
is limited to liver and skeletal muscle. It is also
apparent that despite high levels in the mammary gland and
progestin-responsive breast cancer cells, PRG1 expression is not
confined to progestin target tissues, where, in fact, expression can be
low, e.g. in uterus. Functional analysis of PRG1 protein
will be necessary to determine whether the proteins produced in the
breast, breast tumors, and other tissues have a similar function and
whether PRG1 is the homolog of isoforms identified in other species or
has unique properties.
The progestin regulation of PRG1 mRNA was studied in some detail, and
Northern analysis of PRG1 mRNA levels over a 24-h time period showed a
rapid and transient induction by ORG 2058 that peaked at 3 h and
returned to control levels by 12 h. Several lines of evidence are
consistent with the view that this response is mediated by the PR.
First, the induction of PRG1 mRNA occurred in the presence of three
other synthetic progestins, R5020, MPA, and livial. Second, although
these progestins potentially have some cross-reactivity with the GR
(50, 51), the progestin induction does not appear to be mediated by
this receptor in T-47D or MCF-7 cells as the GR is nonfunctional in
these lines. There was also no evidence for the involvement of AR in
mediating progestin induction. Third, the progestin antagonist RU 486
inhibited the progestin induction of PRG1 mRNA. In MDA-MB-231 cells
expressing high levels of functional GR (39), PRG1 was not strongly
glucocorticoid-regulated, i.e. dexamethasone caused only a
moderate decrease in PRG1 mRNA levels. Interestingly, ORG 2058 had a
similar but weaker effect in this cell line. The mechanism by which
this occurs is not known but does not appear to involve
cross-reactivity of ORG 2058 with the GR.
The progestin induction of PRG1 mRNA was not prevented by the presence
of the protein synthesis inhibitor cycloheximide but was blocked by the
transcription inhibitor actinomycin D. This strongly suggests that
induction of PRG1 by progestin is a transcriptional effect of
ligand-activated PR on the PRG1 gene. A computer search of DNA sequence
from the cosmid clone CRI-JC2015, encompassing 3000 bp from the
initiating methionine in a 5' direction, for an optimal PRE sequence as
determined by in vitro studies (52), revealed several
PRE-like sequences. The PR could be acting by classic mechanisms on
these sequences, i.e. by direct binding, or indirectly, for
example through protein-protein interactions with other transcription
factors. Identification of the precise mechanism involved in progestin
regulation of this gene will require cloning of the PRG1 promoter and
characterization of the putative PREs or other DNA-binding motifs
responsible for progestin induction. This will enable comparisons to be
made with recognized PREs (52, 53, 54, 55) and provide further insight into the
nature of these elements.
PFK-2/FBPase-2 is a bifunctional enzyme that catalyzes the
synthesis and degradation of fructose-2,6-bisphosphate, a molecule that
is a potent stimulator of 6-phosphofructo-1-kinase (PFK-1) and
therefore has a role in control of glycolysis (reviewed in Ref.56).
The tentative assignment of a functional role for PRG1 as a
PFK-2/FBPase-2 isoform suggests a link between progestin effects and
glycolytic control. Because PRG1 induction by progestins precedes
increases in the S phase fraction of cell populations, it is possible
that PRG1 has a role in glycolytic control during the initiation of
cell cycle progression. There is some evidence of such a role for
PFK-2/FBPase-2 under these conditions. An important metabolic response
to growth stimulation by mitogens is an increase in glucose
utilization, suggesting that glycolytic rates are regulated to
coordinate with DNA synthesis (e.g. Refs. 5760).
Furthermore the rat F-type mRNA of PFK-2/FBPase-2 is induced around the
time of the G1/S transition in Rat-1 fibroblasts after
epidermal growth factor or serum stimulation of quiescent cells
(61).
In summary, this study reports the identification and cloning of
PRG1, a new human gene with strong sequence homology to PFK-2/FBPase-2,
thus identifying a third human gene for this enzyme. PRG1 is
transcriptionally regulated by progestins, with induction peaking at
3 h. This induction is not prevented by cycloheximide, and
therefore PRG1 is one of the few human genes demonstrated to be
directly regulated by the PR. This gene may prove to be important in
advancing our understanding of progestin effects on cell growth and
differentiation and gene transcription. Studies are currently underway
to investigate the functional activity of the protein product of PRG1
and its role in progestin action in human breast cancer cell lines.
 |
MATERIALS AND METHODS
|
---|
Reagents
Steroids and growth factors were obtained from the following
sources: ORG 2058, Amersham Australia (Castle Hill, Australia); R5020,
Du Pont Ltd, North Ryde, Australia; MPA, Dr. Dudley Jacobs of Upjohn
Pty Ltd, Sydney, Australia; livial, Dr. Willem Schoonen of Organon
International (Oss, The Netherlands); RU 486, Dr. John-Pierre Raynaud
of Roussel-Uclaf (Romainville, France); 5
-dihydrotestosterone
(5
-androstan-17ß-ol-3-one) and dexamethasone, Sigma Chemical Co.
(St. Louis, MO); hydroxyflutamide, SCH16423, Schering Corp.
(Kenilworth, NJ); human transferrin, Sigma Chemical Co.; human insulin,
Actrapid, CSL-Novo, North Rocks, Australia. Steroids were stored at
-20 C as 1000-fold-concentrated stock solutions in absolute ethanol.
Cycloheximide (Calbiochem-Behring Corp., La Jolla, CA) was dissolved at
20 mg/ml in water and filter sterilized. Actinomycin D (Cosmegen, Merck
Sharp and Dohme Research Pharmaceuticals, Rahway, NJ) was dissolved at
0.5 mg/ml in sterile water and used immediately. Tissue culture
reagents were purchased from standard sources.
Cell Culture
The sources and maintenance of the human breast cancer cell
lines used in this study were as described previously (62). 184 and
184B5 normal breast epithelial cells were the kind gift of Dr. M.
Stampfer (University of California, Berkeley, CA) and were maintained
in mammary epithelial growth medium (Clonetics, San Diego, CA). Tissue
culture experiments in serum-free medium were performed as previously
described (6, 14). Briefly, T-47D cells were taken from stock cultures
and passaged for 6 days in phenol red-free RPMI medium supplemented
with 10% charcoal-treated FCS (6, 14). During this time the cells
received two changes of medium at 1- to 3-day intervals. The cells were
replated into replicate 150-cm2 flasks in medium containing
10% charcoal-treated FCS, and the medium was replaced with serum-free
medium on the next 2 days. Serum-free medium was supplemented with 300
nM human transferrin. In experiments involving ORG 2058,
the final serum-free medium contained 10 µg/ml (1.7 µM)
human insulin. Three days after completion of these pretreatments,
steroid, steroid antagonist, or cycloheximide were added. Control
flasks received vehicle to the same final concentration. Cell cycle
phase distribution was determined by analytical DNA flow cytometry, as
previously described (6, 63). Tissue culture experiments in
serum-containing medium were performed as previously described (63).
The experiments with livial or ORG 2058 and actinomycin D were as for
experiments in serum-containing medium except that the medium contained
5% charcoal-treated FCS. Actinomycin D was added at the same time as
livial or ORG 2058.
RNA Isolation and Northern Analysis
Cells harvested from triplicate 150-cm2 flasks were
pooled and RNA extracted by a guanidinium-isothiocyanate-cesium
chloride procedure, and Northern analysis was performed as previously
described with 20 µg total RNA per lane (6, 11) The membranes were
hybridized overnight (50 C) with probes labeled with
[
-32P]dCTP (Amersham Australia Pty Ltd, Castle Hill,
Australia) using the Random Prime Labeling Kit (Promega, Sydney,
Australia). The membranes were washed at a highest stringency of
0.2 x SSC (30 mM NaCl, 3 mM sodium
citrate [pH 7.0]) + 1% SDS at 65 C and exposed to Kodak X-OMAT or
BIOMAX film at -70 C. Human multiple tissue Northern blots and Master
dot blots (Clontech Laboratories Inc, Palo Alto, CA) were hybridized
under conditions recommended by the manufacturer. The mRNA abundance
was quantitated by densitometric analysis of autoradiographs using
Molecular Dynamics Densitometer and software (Molecular Dynamics,
Sunnyvale, CA). The accuracy of loading was estimated by hybridizing
membranes with a [
-32P]ATP end-labeled oligonucleotide
complementary to 18S ribosomal RNA (rRNA) (39, 64).
Transient Transfection with pMSG-CAT
Transfections of MDA-MB-231 cells with pMSG-CAT (AMRAD Pharmacia
Biotech, Melbourne, Australia) were carried out as previously described
(43) with 40 µg plasmid per 150-cm2 flask without
glycerol shock. Cells were treated with dexamethasone (100
nM) or ORG 2058 (10 nM) with or without RU 486
(100 nM) for 48 h before harvesting for CAT assays
(43).
Differential Display
Differential display was carried out as described (65). Total
RNA, 200 ng, obtained from T-47D cells treated with the synthetic
progestin ORG 2058 for 3 h or from T-47D cells treated with
ethanol control was reverse transcribed with 5'-T12GG as
the primer. The cDNA products were amplified by the PCR using
5'-T12GG and 5'-CAAACGTCGG primers. The PCR products were
separated on a 6% polyacrylamide denaturing sequencing gel. The PCR
product of interest was excised from the gel, reamplified by PCR, and
cloned into the pGEM-T vector (Promega, Madison, WI). DNA sequencing
was performed by the dideoxy chain termination method using T7 DNA
polymerase (AMRAD Pharmacia Biotech) and Sequenase 2.0 kit (Bresatec,
Adelaide, Australia) or by cycle sequencing using the
fmol® DNA Cycle Sequencing System (Promega).
Sequence database searches were performed at the National Center for
Biotechnology Information (NCBI) using the Basic Local Alignment Search
Tool (BLAST) network service.
Library Screening
To obtain additional cDNA sequence,
cDNA libraries derived
from human kidney (Clontech; 2.85 x 105 pfu) and
human heart (Stratagene, La Jolla, CA; 6 x 105 pfu)
were screened using the 32P-labeled differential display
cDNA fragment as a probe under stringent hybridization conditions.
Seven strongly hybridizing clones were isolated and excised for
sequencing using bacterial strain XL1-Blue. Sequencing was performed as
described above. Amino acid sequence alignments were performed using
the computer programs MacVector 4.5.3 (Eastman Kodak Co., Rochester,
NY) and SeqVu (Garvan Institute of Medical Research, Sydney,
Australia).
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to acknowledge members of the Cancer
Research Program for assistance with these studies including Andrea
Brady, Chris Lee, Kimberley Sweeney, Fiona Hammond, Amanda Russell, and
members of the Neurobiology Research Program, Yvonne Hort, Herbert
Herzog, and John Shine, for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Colin K. W. Watts, Garvan Institute of Medical Research, St. Vincents Hospital, Sydney, New South Wales 2010, Australia.
This work was supported by the National Health and Medical Research
Council of Australia (NHMRC) and the Kathleen Cuningham Foundation for
Breast Cancer Research.
1 The nucleotide sequence data reported in this
paper appears in the GSDB and NCBI nucleotide databases with the
accession numbers GSDB:S:75650 and L77662. PRG1 has been assigned the
gene symbol PFKFB3. 
Received for publication October 1, 1996.
Revision received December 30, 1996.
Accepted for publication January 15, 1997.
 |
REFERENCES
|
---|
-
Clarke CL, Sutherland RL 1990a Progestin regulation of
cellular proliferation. Endocr Rev 11:266301
-
Shi YE, Liu YE, Lippman ME, Dickson RB 1994 Progestins and
anti-progestins in mammary tumour growth and metastasis. Hum Reprod
9[Suppl]1:162173
-
Anderson TJ, Battersby S, King RJB, McPherson K, Going JJ 1989 Oral contraceptive use influences resting breast proliferation.
Hum Pathol 20:11391144[Medline]
-
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery
Jr CA, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit pleiotropic reproductive abnormalities.
Gene Dev 9:22662278[Abstract]
-
Sedlacek SM, Horwitz KB 1984 The role of progestins and
progesterone receptors in the treatment of breast cancer. Steroids 44:46784[Medline]
-
Musgrove EA, Lee CSL, Sutherland RL 1991 Progestins both
stimulate and inhibit breast cancer cell cycle progression while
increasing expression of transforming growth factor
, epidermal
growth factor receptor, c-fos, and c-myc genes.
Mol Cell Biol 11:50325043[Medline]
-
Alkhalaf M, Murphy LC 1992 Regulation of c-jun and
jun-B by progestins in T-47D human breast cancer cells. Mol
Endocrinol 6:16251633[Abstract]
-
Murphy LC, Alkhalaf M, Dotzlaw H, Coutts A, Haddad-Alkhalaf B 1994 Regulation of gene expression in T-47D human breast cancer cells
by progestins and anti-progestins. Hum Reprod 9[Suppl]1:174180
-
Joyeux C, Rochefort H, Chalbos D 1989 Progestin increases
gene transcription and messenger ribonucleic acid stability of fatty
acid synthetase in breast cancer cells. Mol Endocrinol 4:681686
-
Misrahi M, Loosfelt H, Atger M, Meriel C, Zerah V, Dessen
P, Milgrom E 1988 Organisation of the entire rabbit progesterone
receptor mRNA and of the promoter and 5' flanking region of the gene.
Nucleic Acids Res 16:54595472[Abstract]
-
Alexander IE, Clarke CL, Shine J, Sutherland RL 1989 Progestin
inhibition of progesterone receptor gene expression in human breast
cancer cells. Mol Endocrinol 3:13771386[Abstract]
-
Jantzen K, Fritton HP, Igo-Kemenes T, Espel E, Janich S, Cato
AC, Mugele K, Beato M 1987 Partial overlapping of binding sequences for
steroid hormone receptors and DNaseI hypersensitive sites in the rabbit
uteroglobin gene region. Nucleic Acids Res 11:45354552
-
Theveny B, Bailly A, Rauch C, Rauch M, Delain E, Milgrom E 1987 Association of DNA-bound progesterone receptors. Nature 329:7981[CrossRef][Medline]
-
Musgrove EA, Hamilton JA, Lee CSL, Sweeney KJE, Watts CKW,
Sutherland RL 1993 Growth factor, steroid, and steroid antagonist
regulation of cyclin gene expression associated with changes in T-47D
human breast cancer cell cycle progression. Mol Cell Biol 13:35773587[Abstract]
-
Alexander IE, Shine J, Sutherland RL 1990 Progestin regulation
of estrogen receptor messenger RNA in human breast cancer cells. Mol
Endocrinol 4:821828[Abstract]
-
Roman SD, Clarke CL, Hall RE, Alexander IE, Sutherland RL 1992 Expression and regulation of retinoic acid receptors in human breast
cancer cells. Cancer Res 52:22362242[Abstract]
-
Murphy LC, Murphy LJ, Shiu RP 1988 Progestin regulation of
EGF-receptor mRNA accumulation in T-47D human breast cancer cells.
Biochem Biophys Res Commun 150:1926[Medline]
-
Ormandy CJ, Graham J, Kelly PA, Clarke CL, Sutherland RL 1992 The effect of progestin on prolactin receptor gene transcription in
human breast cancer cells. DNA Cell Biol 11:721726[Medline]
-
Papa V, Hartmann KK, Rosenthal SM, Maddux BA, Siiteri PK,
Goldfine ID 1991 Progestins induce down-regulation of insulin-like
growth factor-I (IGF-I) receptors in human breast cancer cells:
potential autocrine role of IGF-II. Mol Endocrinol 5:709717[Abstract]
-
Papa V, Reese CC, Brunetti A, Vigneri R, Siiteri PK, Goldfine
ID 1990 Progestins increase insulin receptor content and insulin
stimulation of growth in human breast carcinoma cells. Cancer Res 50:78587862[Abstract]
-
Murphy LC, Murphy LJ, Dubik D, Bell GI, Shiu RP 1988 Epidermal
growth factor gene expression in human breast cancer cells: regulation
of expression by progestins. Cancer Res 48:45554560[Abstract]
-
Murphy LC, Dotzlaw H 1989 Regulation of transforming growth
factor alpha and transforming growth factor beta messenger ribonucleic
acid abundance in T-47D, human breast cancer cells. Mol Endocrinol 3:6117[Abstract]
-
Gong Y, Anzai Y, Murphy LC, Ballejo G, Holinka CF, Gurpide E,
Murphy LJ 1991 Transforming growth factor gene expression in human
endometrial adenocarcinoma cells: regulation by progestins. Cancer Res 51:547681[Abstract]
-
Poutanen M, Isomaa V, Kainulainen K, Vihko R 1990 Progestin
induction of 17 beta-hydroxysteroid dehydrogenase enzyme protein in the
T-47D human breast-cancer cell line. Int J Cancer 46:897901[Medline]
-
Coutts A, Murphy LJ, Murphy LC 1994 Expression of insulin-like
growth factor binding proteins by T-47D human breast cancer cells:
regulation by progestins and anti-estrogens. Breast Cancer Res Treat 32:153164[Medline]
-
Di Lorenzo D, Albetini A, Zava D 1991 Progestin regulation of
alkaline phosphatase in the human breast cancer cell line T47D. Cancer
Res 51:44704475[Abstract]
-
Hagley RD, Hissom JR, Moore MR 1987 Progestin stimulation of
lactate dehydrogenase in the human breast cancer cell line T-47D.
Biochim Biophys Acta 930:167172[CrossRef][Medline]
-
Liang P, Pardee AB 1992 Differential display of eukaryotic
messenger RNA by means of the polymerase chain reaction. Science 257:967971[Medline]
-
Zheng C-J, Ma NS-F, Dorman TE, Wang M-T, Braunschweiger K,
Soares L, Schuster MK, Rothschild CB, Bowden DW, Torrey D, Keith TP,
Moir DT, Mao J-I 1994 Development of 124 sequence-tagged sites and
cytogenetic localization of 217 cosmids for human chromosome 10.
Genomics 22:5567[CrossRef][Medline]
-
Stryer L 1988 Flow of genetic information. In: Biochemistry,
ed 3. W.H. Freeman and Co, New York, pp 110112
-
Lange AJ, Pilkis SJ 1990 Sequence of human liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Nucleic Acids Res 18:3652[Medline]
-
Ventura F, Ambrosio S, Bartrons R, El-Maghrabi MR, Lange AJ,
Pilkis SJ 1995 Cloning and expression of a catalytic core bovine brain
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem Biophys
Res Commun 209:11401148[CrossRef][Medline]
-
Sakata J, Uyeda K 1990 Bovine heart fructose 6-phosphate,
2-kinase/fructose 2,6-bisphosphatase: complete amino acid sequence and
localization of phosphlorylation sites. Proc Natl Acad Sci USA 87:49514955[Abstract]
-
Kozak M 1987 An analysis of 5'-noncoding sequences from 699
vertebrate messenger RNAs. Nucleic Acids Res 15:81258132[Abstract]
-
Lively MO, El-Maghrabi MR, Pilkis J, DAngelo G, Colosia AD,
Ciavola JA, Fraser BA, Pilkis SJ 1988 Complete amino acid sequence of
rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
J Biol Chem 263:839849[Abstract/Free Full Text]
-
Sakata J, Abe Y, Uyeda K 1991 Molecular cloning of the DNA and
expression and characterization of rat testes
fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. J Biol
Chem 266:1576415770[Abstract/Free Full Text]
-
Kitamura K, Kangawa K, Matsuo H, Uyeda K 1988 Phosphorylation
of myocardial fructose-6-phosphate,2-kinase:
fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and
protein kinase C. Activation by phosphorylation and amino acid
sequences of the phosphorylation sites. J Biol Chem 263:1679616801[Abstract/Free Full Text]
-
Pilkis SJ, Claus TH, Kurland IJ, Lange AJ 1995 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic
signaling enzyme. Annu Rev Biochem 64:799835[CrossRef][Medline]
-
Hall RE, Lee CSL, Alexander IE, Shine J, Clarke CL, Sutherland
RS 1990 Steroid hormone receptor gene expression in human breast cancer
cells: inverse relationship between oestrogen and glucocorticoid
receptor messenger RNA levels. Int J Cancer 46:10811087[Medline]
-
Sutherland RL, Hall RE, Pang GYN, Musgrove EA, Clarke CL 1988 Effect of medroxyprogesterone acetate in proliferation and cell cycle
kinetics of human mammary carcinoma cells. Cancer Res 48:50845091[Abstract]
-
Carson-Jurica MA, Schrader WT, OMalley BW 1990 Steroid
receptor family: structure and functions. Endocr Rev 11:201220[Abstract]
-
Ewing TM, Murphy LJ, Ng M-L, Pang GYN, Lee CSL, Watts CKW,
Sutherland RL 1989 Regulation of epidermal growth factor receptor by
progestins and glucocorticoids in human breast cancer cell lines. Int J
Cancer 44:744752[Medline]
-
Clarke CL, Graham J, Roman SD, Sutherland RL 1991 Direct
transcriptional regulation of the progesterone receptor by retinoic
acid diminishes progestin responsiveness in the breast cancer cell line
T-47D. J Biol Chem 266:1896918975[Abstract/Free Full Text]
-
Bardon S, Vignon F, Chalbos D, Rochefort H 1985 RU486, A
progestin and glucocorticoid antagonist, inhibits the growth of breast
cancer cells via the progesterone receptor. J Clin Endocrinol
Metab 60:692697[Abstract]
-
Rochefort H, Chalbos D 1984 Progestin-specific markers in
human cell lines: biological and pharmacological applications. Mol Cell
Endocrinol 36:310[CrossRef][Medline]
-
Elder PK, Schmidt LJ, Ono T, Getz MJ 1984 Specific stimulation
of actin gene transcription by epidermal growth factor and
cycloheximide. Proc Natl Acad Sci USA 81:74767480[Abstract]
-
Greenberg ME, Ziff EB 1984 Stimulation of 3T3 cells induces
transcription of the c-fos proto-oncogene. Nature 311:433438[Medline]
-
Greenberg ME, Hermanowski AL, Ziff EB 1986 Effect of protein
synthesis inhibitors on growth factor activation of c-fos,
c-myc, and actin gene transcription. Mol Cell Biol 6:10501057[Medline]
-
Hilliker CE, Darville MI, Aly MS, Chikri M, Szpirer C,
Marynen P, Rousseau GG, Cassiman J-J 1991 Human and rat chromosomal
localization two genes for
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of
somatic cell hybrids and in situ hybridization. Genomics 10:867873[Medline]
-
Poulin R, Baker D, Poirier D, Labrie F 1989 Androgen and
glucocorticoid receptor-mediated inhibition of cell proliferation by
medroxyprogesterone acetate in ZR-751 human breast cancer cells.
Breast Cancer Res Treat 13:161172[Medline]
-
Poulin R, Baker D, Poirier D, Labrie F 1990 Multiple actions
of synthetic progestins on the growth of ZR-751 human breast
cancer cells: An in vitro model for the simultaneous assay
of androgen, progestin, estrogen, and glucocorticoid agonistic and
antagonistic activities of steroids. Breast Cancer Res Treat 17:197210
-
Lieberman BA, Bona BJ, Edwards DP, Nordeen SK 1993 The
constitution of a progesterone response element. Mol Endocrinol 7:515527[Abstract]
-
Slater EP, Cato AC, Karin M, Baxter JD, Beato M 1988 Progesterone induction of metallothionein-IIA gene expression. Mol
Endocrinol 2:485491[Abstract]
-
Cassady AI, King AG, Cross NC, Hume DA 1993 Isolation and
characterization of the genes encoding mouse and human type-5 acid
phosphatase. Gene 130:201207[CrossRef][Medline]
-
Lamian V, Gonzalez BY, Michel FJ, Simmen RC 1993 Non-consensus
progesterone response elements mediate the progesterone-regulated
endometrial expression of the uteroferrin gene. J Steroid Biochem Mol
Biol 46:439450[CrossRef][Medline]
-
Hue L, Rousseau GG 1993 Fructose 2,6-bisphosphate and the
control of glycolysis by growth factors, tumor promoters and oncogenes.
Adv Enzyme Regul 33:97110[CrossRef][Medline]
-
Diamond I, Legg A, Schneider JA, Rozengurt E 1978 Glycolysis
in quiescent cultures of 3T3 cells. Stimulation by serum, epidermal
growth factor, and insulin in intact cells and persistence of the
stimulation after cell homogenization. J Biol Chem 253:866871[Abstract]
-
Bruni P, Farnararo M, Vasta V, DAlessandro A 1983 Increase
of the glycolytic rate in human resting fibroblasts following serum
stimulation. The possible role of the fructose-2,6-bisphosphate. FEBS
Lett 159:3942[CrossRef][Medline]
-
Bosca L, Rousseau GG, Hue L 1985 Phorbol 12-myristate
13-acetate and insulin increase the concentration of fructose
2,6-bisphosphate and stimulate glycolysis in chicken embryo
fibroblasts. Proc Natl Acad Sci USA 82:64406444[Abstract]
-
Marjanovic S, Skog S, Heiden T, Tribukait B, Nelson BD 1991 Expression of glycolytic isoenzymes in activated human peripheral
lymphocytes: cell cycle analysis using flow cytometry. Exp Cell Res 193:425431[Medline]
-
Darville MI, Antoine IV, Mertens-Strijthagen JR, Dupriez VJ,
Rousseau GG 1995 An E2F-dependent late-serum-response promoter in a
gene that controls glycolysis. Oncogene 11:15091517[Medline]
-
Buckley MF, Sweeney KJE, Hamilton JA, Sini RL, Manning DL,
Nicholson RI, deFazio A, Watts CK, Musgrove EA, Sutherland RL 1993 Expression and amplification of cyclin genes in human breast
cancer. Oncogene 8:212733[Medline]
-
Musgrove EA, Wakeling AE, Sutherland RL 1989 Points of action
of estrogen antagonists and a calmodulin antagonist within the MCF-7
human breast cancer cell cycle. Cancer Res 49:23982404[Abstract]
-
Chan Y-L, Guttell R, Noller HF, Wool IG 1984 The nucleotide
sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal
for the secondary structure of 18 S ribosomal ribonucleic acid. J
Biol Chem 259:224230[Abstract/Free Full Text]
-
Liang P, Averboukh L, Keyomarsi K, Sager R, Pardee AB 1992 Differential display and cloning of messenger RNAs from human breast
cancer vs. mammary epithelial cells. Cancer Res 52:69666968[Abstract]
-
Sakai A, Kato M, Fukasawa M, Ishiguro M, Furuya E, Sakakibara
R 1996 Cloning of cDNA encoding for a novel isozyme of
fructose-6-phosphate,2-kinase/fructose 2,6-bisphosphatase from human
placenta. J Biochem 119:506511[Abstract]
-
Joaquin M, Salvado C, Bellosillo B, Lange AJ, Gil J, Tauler A 1997 Effect of growth factors on the expression of 6-phosphofructo
2-kinase/fructose-2,6-bisphosphatase in rat-1 fibroblasts. J Biol Chem 27:28462851[CrossRef]