Molecular Cloning of TA16, a Transcriptional Repressor That May Mediate Glucocorticoid-Induced Growth Arrest of Leiomyosarcoma Cells
Weimin Fan,
Jian-Xing Ma,
Lirong Cheng and
James S. Norris
Departments of Pathology and Laboratory Medicine (W.F., L.C.),
Ophthalmology (J.M.), Microbiology and Immunology (J.S.N), and Medicine
(W.F., J.S.N.) Medical University of South Carolina Charleston,
South Carolina 29425
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ABSTRACT
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The DDT1 MF2 smooth muscle
tumor cell line was derived from an estrogen/androgen-induced
leiomyosarcoma that arose in the ductus deferens of a Syrian hamster.
The growth of this cell line is arrested at the G0/G1 phase of the cell
cycle after treatment with glucocorticoids. To identify the putative
gene(s) that are potentially involved in this hormone-induced cell
growth arrest, we have used a differential screening technique to clone
those genes whose expression is induced or up-regulated by
glucocorticoids. A number of glucocorticoid response genes were thereby
isolated from the leiomyosarcoma cells. One of these clones, termed
TA16, was found to be markedly up-regulated by glucocorticoids in
DDT1 MF2 cells, but only marginally changed in
GR1 cells, a glucocorticoid-resistant variant that was selected from
the wild type DDT1 MF2 cell. Isolation and
sequencing of its intact cDNA indicated that the TA16 encodes a protein
485 amino acids long, and its sequence is closely homologous to a novel
transcriptional repressor that presumably represses the transcription
activity of some zinc finger transcriptional factors through a direct
interaction. Transfection assays demonstrated that introduction of an
antisense TA16 cDNA expression vector, controlled by an MMTV promoter,
into the DDT1 MF2 cell significantly relieved
the glucocorticoid-induced cell growth arrest. This finding suggests
that TA16 might participate in the mediation of glucocorticoid-induced
cell cycle arrest in leiomyosarcoma cells.
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INTRODUCTION
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The DDT1 MF2 smooth muscle tumor cell line was derived
from an estrogen/androgen-induced leiomyosarcoma that arose in the
ductus deferens of a Syrian hamster (1). This cell line expresses
receptors for both androgens and glucocorticoids, and its proliferation
is differentially sensitive to these classes of steroid hormones.
Androgens can stimulate DDT1 MF2 cell growth and augment
the level of intracellular androgen receptors (2, 3, 4), whereas
glucocorticoids induced cell growth arrest at the G0/G1 phase of the
cell cycle (5, 6). Through transplantation of DDT1 MF2
cells into Syrian hamsters (7), the in vivo inhibitory
action of glucocorticoids on tumor growth was also observed if the
hamster host was implanted with silastic tubing containing crystalline
triamcinolone acetonide. In addition, by genetic selection, a
glucocorticoid-resistant variant cell line DDT1 MF2 GR1 was
developed from the wild type leiomyosarcoma cells. The growth of GR1
cells is not sensitive to the inhibitory action of glucocorticoids (8, 9), although recent studies demonstrated that some other glucocorticoid
responses, both primary and secondary, may still exist in the GR1 cells
(10).
Glucocorticoids, a class of steroid hormones, play a fundamental role
in the physiological, proliferative, and developmental control of
animal tissue by regulating a diversity of gene networks and cellular
processes (11, 12). Treatment with glucocorticoids has been found to
inhibit in vivo and in vitro growth of many types
of normal and transformed cells (13, 14, 15, 16, 17, 18, 19). In the past few years, in
addition to the DDT1 MF2 cell line, several other tumor
cells, such as the prostate tumor-derived cell line A3327H-G8-A1 (18, 20) and the mammary carcinoma-derived cell line Con8, were also found
to arrest their growth in response to glucocorticoids in cell growth
arrest (21, 22). Moreover, the inhibitory action of glucocorticoids in
these tumor cells seems to share many common features. For example, all
three of these tumor cells are arrested by glucocorticoids at the G0/G1
phase of the cell cycle (5, 18, 23). Also, glucocorticoid-induced
growth suppression in these tumor cells is reversible. Thus, cells
return to normal growth and cell cycle progression after removal of the
steroid (5, 10, 23). These common features suggest that a common
signaling pathway may exist by which glucocorticoids arrest cell
growth.
The mechanism underlying glucocorticoid-mediated growth suppression in
tumor cells is not entirely clear, but requires functional
glucocorticoid receptors, an appropriate hormone environment, and, most
likely, changes in the expression of growth-stimulatory or
growth-inhibitory factors (5, 10, 24, 25). Previous studies have shown
that glucocorticoids, after binding to their cognate receptor, can have
either positive or negative effects on gene transcription (11, 26). The
direction of regulation depends upon both protein-protein interaction,
such as with the c-fos/jun transcription complex (27) and a
specific DNA recognition sequence within the upstream regulatory region
(28). Therefore, one possibility for the antiproliferative effects of
glucocorticoids on tumor cell growth is that they induce or activate
growth suppressor(s) or negative growth-regulatory protein(s), which
either prolong the current cell cycle or prevent cells from entering
into the next stage of the cell cycle. Early studies in our laboratory
have demonstrated that glucocorticoid-induced cell growth arrest in
DDT1 MF2 cells could be abrogated by a low concentration of
cycloheximide (1 µg/ml) (6), suggesting that ongoing protein
synthesis is required for glucocorticoid-mediated cell growth
suppression. In addition, Webster et al recently reported
that, by fusing glucocorticoid-suppressible Con8 cells with either
8RUV7 growth suppression-resistant variant or rat hepatoma cells that
constitutively proliferate in the presence of glucocorticoids, the
glucocorticoid-suppressible phenotype of Con8 tumor cells was dominant.
This suggests the existence of intracellular regulatory factors, under
glucocorticoid control, that function as trans-acting
suppressors of tumor cell growth. Therefore, we hypothesize that a
putative tumor suppressor gene or a negative growth-regulatory factor
might be induced or up-regulated by glucocorticoids and, in turn,
affect tumor cell growth. Based on this hypothesis, a feasible approach
to identification of the putative regulatory factor(s) was first to
clone those genes whose expression is induced or regulated by
glucocorticoids.
In the present study, we have used a PCR-mediated differential
screening technique (29) to isolate those genes whose corresponding
mRNAs are transcriptionally regulated by glucocorticoids. A number of
glucocorticoid response genes were thereby isolated. One of those
genes, termed TA16, was found to be markedly induced by glucocorticoids
in the wild type DDT1 MF2 cells, but only slightly changed
in the glucocorticoid-resistant variant GR1 cell. Further
characterization of this gene through cloning and sequencing its
full-length cDNA indicated that TA16 encodes a protein closely related
to NAB1 (NGFI-A-binding protein), a novel transcriptional repressor,
which was recently isolated by another group through the yeast
two-hybrid cloning system by using zinc finger transcriptional factor
NGFI-A as a target bait (30). Because this novel class of transcription
repressor was assumed to repress transcriptional activation mediated by
NGFI-A and several other zinc finger transcriptional factors involved
in the regulation of many cell growth factors or cell cycle-related
proteins (30, 31), the high homology of TA16 to NAB1 suggests that the
induction of TA16 by glucocorticoids might be related to the mediation
of glucocorticoid-induced cell growth arrest. Therefore, after the
isolation of intact TA16 cDNA, transfection assays were conducted to
examine the possible involvement of TA16 in the signaling transduction
by which glucocorticoids arrest cell growth. As a result, we found that
introduction of an antisense TA16 expression vector could markedly
relieve the cell growth arrest due to glucocorticoids. This finding
suggests that TA16 may participate in the mediation of
glucocorticoid-induced growth arrest in DDT1 MF2
leiomyosarcoma cells.
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RESULTS
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Initial Cloning of TA16 by mRNA Differential Display
Total RNAs isolated from glucocorticoid-treated
[(10-7 M triamcinolone acetonide (TA)] or
untreated DDT1 MF2 cells were subjected to mRNA
differential display (29). For each group, 40 different combinations of
primer sets made of four degenerate oligo(dT) primers, T12
MG, T12 MA, T12 MT, or T12 MC and
ten arbitrary 10 oligomers (AP110) were used to obtain differential
displays. The reactions showing uniquely or differentially expressed
bands in the group treated with glucocorticoids were repeated to ensure
reproducibility. The cDNA fragments found to be reproducibly induced or
regulated by glucocorticoids were excised from dried acrylamide gels,
reamplified by PCR, subcloned into pCR II vectors (Invitrogen, San
Diego, CA), and sequenced. Initially, more than two dozen
glucocorticoid response genes were isolated by this process. Most of
these cloned cDNA fragments belonged to novel genes (32). Northern
blots confirmed that about one third of these genes were clearly
induced or regulated by glucocorticoids. The remaining clones were
either too weak to detect or were false positives (data not shown).
Clone TA16 was one of the cloned cDNA fragments induced by
glucocorticoids and consisted of 286 bp. Northern blotting indicated
that the expression of TA16 in the DDT1 MF2 cells was
markedly induced by glucocorticoids (10-7 M
TA), but only marginally changed in the GR1 cell, a
glucocorticoid-resistant variant cell line (see Fig. 1
).

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Figure 1. Messenger RNA Differential Display and Northern
Blots
Left, Differential mRNA display. Total RNA was isolated
from DDT1 MF2 cells treated or untreated with
glucocorticoid (10-7 M TA). The RNA was
reverse transcribed and followed by PCR in the presence of
[35S]dATP (see Materials and Methods for
details). The PCR products were then displayed in a 6% sequence gel
and autoradiographed. The arrow indicates the fragment
representing TA16. This cDNA fragment was reamplified by PCR and
subcloned into pCR II vector. Right, Northern blotting.
Total RNA isolated from DDT1 MF2 and GR1 cells untreated or
treated with glucocorticoids (10-7 M TA) for
24 h. RNA (20 µg/lane) was size-fractionated by
formaldehyde/agarose gel electrophoresis. The subcloned TA16 cDNA
fragment and hamster ß-actin were used as a probes for the Northern
analysis.
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Isolation and Sequencing of Intact cDNA of TA16
Due to its significant induction by glucocorticoids, Clone TA16
was selected with several other cloned cDNA fragments to screen a cDNA
library that was constructed with mRNA isolated from
glucocorticoid-treated DDT1 MF2 cells (33). As depicted in
Fig. 2
, the 286-bp cDNA fragment of TA16 was used as the
probe for the screening. After three cycles of screening, two different
cDNA clones, coded as TA16-A2 and TA16-F2, were isolated. DNA
sequencing indicated that both clones essentially shared the same
sequence, but TA16-A2 was 175 nucleotides longer in the 5'-coding
region. Also, for unknown reasons, the 3'-end of Clone TA16-A2 was 15
bp longer than the TA16-F2, although both have poly (A) tails (see Fig. 2
). However, sequence analysis indicated that both clones apparently
lack part of the 5'-coding sequence. We repeated the library screening
using clone TA16-A2 as a probe, but failed to get a full-length cDNA
clone. The missing 5'-coding region of TA16 was subsequently isolated
by the 5'-RACE cloning technique (34). DNA sequencing indicated that
the full-length TA16 cDNA consists of 2389 nucleotides with one open
reading frame that encodes a protein 485 amino acids long (Fig. 3
). A subsequent computer search revealed that TA16
shows high homology to NAB1, a novel transcription repressor, which was
recently isolated by another group (30). Because its protein sequence
only differs by 15 to 20 amino acids from the NAB1 derived from other
species (Fig. 4
), TA16 was believed to represent this
class of transcriptional repressor in hamster.

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Figure 2. Cloning Strategy and Structural Features of
Full-Length TA16 cDNA
Step 1: the TA16 cDNA fragment cloned by mRNA differential display was
used as a probe to screen a hamster cZAP cDNA library. Two incomplete
cDNA clones, SK-TA16-A2 and SK-TA16-F2, were thereby isolated, which
are in the Bluescript SK vectors (Strategene, La Jolla, CA); Step 2:
the missing 5'-coding region was isolated by a 5'-RACE cloning
technique as described in Materials and Methods; Step 3:
utilizing the unique XhoI restriction site in the coding
region of TA16 cDNA, two separate pieces of cDNAs were ligated together
and inserted into pCR II vector (PCR II-TA16). Shaded
bar indicates that coding sequence; open bar
indicates the noncoding sequence.
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Figure 3. Nucleotide and Deduced Amino Acid Sequence TA16
The termination codon is underlined and marked as a
star. The putative poly(A)+ tail signal
sequence is also underlined. This cDNA sequence has been
deposited in the GenBank; the assigned access number is U88975.
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Figure 4. Comparison of TA16 Protein Sequence with
Transcriptional Repressor NAB1 Isolated from Human, Mouse, and Rat
Residues that are identical are shaded. The human, mouse
and rat NAB1 sequences were derived from GenBank. Their accession
numbers are U47007, U47008, and U17253, respectively.
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Effect of TA16 on Glucocorticoid-Induced Cell Growth Arrest
To determine whether the induction of TA16 by glucocorticoids is
related to cell growth arrest, TA16 cDNA was inserted into expression
vector pMAMneo-MMTV (CLONTECH, Palo Alto, CA), which was controlled by
a glucocorticoid-inducible promoter (35), either in a sense or
antisense orientation (see Fig. 5
). Subsequently, these
expression vectors were transfected into wild type DDT1 MF2
cells. After stably transfected cell lines were established, the
effects of TA16 on the inhibitory action of glucocorticoids was
evaluated by comparing the growth pattern of the cells with or without
introduction of TA16. Our results indicated that even though the growth
pattern of the cells transfected with sense TA16 expression vectors was
essentially unchanged, i.e like the wild type
DDT1 MF2, growth of these cells was still inhibited by
glucocorticoids (data not shown). However, the cells transfected with
an antisense TA16 were less sensitive to the inhibitory action of
glucocorticoids on cell growth arrest. As shown in Fig. 6
, the growth of wild type DDT1 MF2 cells
and the cells transfected with the vector alone were dramatically
inhibited by glucocorticoids (10-7 M TA), but
glucocorticoid-induced growth arrest in the cells transfected with an
antisense TA16 expression vector was relieved (see Fig. 6
). In
addition, with light microscopy, we also observed that the size of cell
colonies proliferating from single cells were much larger in the group
with the antisense TA16 expression vector when in the presence of
glucocorticoids (see Fig. 7
). These results indicated
that glucocorticoid-induced growth suppression was partly relieved by
transfection with a glucocorticoid-inducible TA16 antisense expression
vector. In addition, to confirm expression and induction of the
antisense TA16 by glucocorticoid in transfected cells, ribonuclease
(RNase) protection assays were performed. As depicted in Fig. 8
, the basal expression of TA16 antisense mRNA was
relatively low (lanes 5, 7, and 9), whereas in the presence of
glucocorticoid (10-7 M TA), the level of
antisense TA16 mRNA was significantly increased in all three
transfected cell lines (lanes 6, 8, and 10).

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Figure 5. Construction of TA16 Expression Vectors
By utilizing the unique restriction sites in the pCR II and MAMneo-MMTV
vectors, partial or entire TA16 cDNA was cut out from pCR II-TA16
vector and subsequently inserted into the MAMneo-MMTV expression
vectors, which are under control of a glucocorticoid-sensitive
promoter. All constructions were confirmed by DNA sequencing.
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Figure 6. Effect of Antisense TA16 on Glucocorticoid-Induced
Cell Growth Arrest
Untransfected DDT1 MF2 cells and the cells transfected with
an antisense TA16 expression vector or vector alone were cultured in
24-well plates at an initial density of 104 cells per well.
TA (10-7 M) or 100% ethanol (1 µl/ml) was
added to the cells, designated as TA or control groups, respectively.
Cell number was counted at the indicated time points using a Coulter
Counter.
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Figure 7. Growth Pattern of DDT1 MF2 Cells with
or without Transfection of Antisense TA16
Cell culture and glucocorticoid treatment was the same as described in
Fig. 6 . Media with or without glucocorticoid (10-7
M TA) were changed every 4 days. On the tenth day of cell
culture, cells were examined and photographed using phase contrast
microscopy. DDT1 MF2 ant3 was a cell line
transfected with an antisense TA16 expression vector.
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Figure 8. RNase Protection Assay
[32P]UTP-labeled RNA transcripts (189 bp) complementary
to antisense TA16 mRNA sequence were synthesized and hybridized with
the RNAs isolated from untransfected DDT1 MF2 or the cells
transfected with antisense TA16 expression vectors as described in
Materials and Methods. After RNase digestion, the
samples were analyzed on a 6% acrylamide gel followed by
autoradiography. Lane 1, Undigested probe. Lane 2, Probes plus yeast
RNA plus nuclease (negative control). Lanes 3 and 4, Untransfected
DDT1 MF2 cells. Lanes 5 and 6, Lanes 7 and 8; Lanes 810
represent three cell lines (TA16-Ant 2, Ant 3, and Ant 6) transfected
with an antisense TA16 expression. Lanes 11 and 12 are the
DDT1 MF2 cells transfected with MAMneo-MMTV vector only. In
addition, lanes 2, 4, 6, 8, 10, and 12 were treated with
10-7 TA for 24 h before the RNAs were isolated.
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DISCUSSION
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We have described the discovery and cloning of TA16, a
transcription repressor, by the mRNA differential display procedure.
The mRNA differential display technique is a powerful tool with which
to identify and isolate the genes that are differentially expressed in
a pair of related cells or under altered conditions (29). Technically,
it was not difficult to identify and clone these genes whose expression
was transcriptionally regulated by glucocorticoids. More than two dozen
glucocorticoid response cDNA fragments were initially isolated through
this strategy (32). However, establishment of gene function related to
glucocorticoid-induced cell growth arrest has been more difficult.
Fortunately, with the glucocorticoid resistance variant cell line (GR1)
in which cell growth is no longer sensitive to glucocorticoids (5, 10),
we were able to compare expression and glucocorticoid induction of
these candidate genes with the wild type DDT1 MF2 cells.
Those genes whose expression was clearly induced by glucocorticoids in
the wild type cells, but were not or only marginally changed in the GR1
cells, were selected for further study and cloning. When the
full-length TA16 cDNA was eventually isolated and sequenced, it was
found to be closely homologous to NAB1, a novel class of transcription
repressor (31). This finding suggested that TA16 may be involved in the
mediation of glucocorticoid-induced cell growth arrest through
modulation of gene transcription.
The novel transcription repressor, NAB1, was initially isolated by
Busso et al. (30) through the yeast two-hybird cloning
system using NGFI-A, a zinc finger transcriptional factor, as a target
bait. The same paper also demonstrated that NAB1 could repress the
transcription activity of a number of zinc finger transcription
factors, such as NGFI-A and Krox20. These closely related zinc finger
transcription factors have very similar DNA-binding domains and
activate transcription from a consensus sequence, 5'-GGG(G/T)GGGCG-3'
(36, 37), present in the promoters of a number of genes encoding growth
factors [platelet-derived growth factor
-chain, insulin-like growth
factor II, and transforming factor ß (38, 39, 40)] and proteins involved
in cell cycle control [c-myc, thymidine kinase, cyclin D,
and c-myb (41, 42, 43)]. Krox20 has also been shown to regulate
Hoxb-2 in the developing hindbrain (44). Because the expression of
these transcription factors has been reported to be induced by a
variety of proliferative, differentiative, and apoptotic stimuli (45, 46), it was generally believed that these zinc finger transcription
factors or activators play important roles in the regulation of cell
growth, differentiation, and even apoptosis. In light of the high
homology of protein sequences between TA16 and NAB1 derived from other
species, TA16 is considered as a member of this class of
transcriptional repressor. Thus, it is highly possible that the
induction of TA16 by glucocorticoids may be related to the
hormone-induced cell growth arrest.
By Northern blots, expression of TA16 was demonstrated to be induced by
glucocorticoids in the DDT1 MF2 cells (see Fig. 1
). If we assume that
this high expression of TA16 is associated with the
glucocorticoid-mediated cell growth arrest, tumor cells should lose
their sensitivity to glucocorticoid-induced growth arrest once TA16 is
selectively suppressed or inactivated. Essentially, the data obtained
from transfection assays confirmed our hypothesis, although the
glucocorticoid-induced growth arrest was only partially relieved
(about 2-fold compared with the wild type cells) in the cells with an
antisense TA16 expression vector (see Figs. 6
and 7
). Considering the
fact that TA16 is highly induced by glucocorticoids, it might be
reasoned that antisense TA16 mRNA could only partially block TA16
protein synthesis. In addition, we have also tried the transfection
assay in the GR1 cell, but neither the sense nor antisense TA16
expression vector could significantly alter the growth pattern of GR1
cells (data not shown). Basically, we were not surprised by these
results because our early studies have suggested that there might be
more than one mutation responsible for the resistance of the GR1 cell
to glucocortidicoid-mediated growth suppression. For example, we have
demonstrated that both mRNA levels and the activity of glucocorticoid
receptors in the GR1 cell were much lower than that in the wild type
DDT1 MF2 cell, which was only about one tenth compared with
the wild type cell (6, 10). Thus, most glucocorticoid response events
occurring in the GR1 cell, including the glucocorticoid-sensitive
murine Moloney transcription virus (MMTV) promotor, are also
significantly lower in the GR1 cell (10). Therefore, it is difficult to
assess the TA16 in the GR1 cell because, even in the
glucocorticoid-sensitive wild type cell, we could only detect partial
relief of glucocorticoid-induced growth arrest through the transfection
assay (see Fig. 6
). On the other hand, due to a lack of specific
antibodies for this class of transcription repressor, at this moment,
TA16 can be only considered an important candidate gene. Further
studies will be necessary to define the possible role of TA16 in this
hormone-mediated cell growth arrest.
Negative regulation of transcription from specific promoters is an
important mechanism for controlling gene expression. One means by which
negative regulators can function is through direct interactions with
transcriptional activators. Examples include the regulation of NF-
B
by its sequestration in the cytoplasm by I
B (47, 48) and the
modulation of E2F activity by its association with the retinoblastoma
gene product pRb (49, 50). If TA16 is indeed involved in
glucocorticoid-induced cell growth arrest, as a member of NAB1
transcription repressor family, it would be predicted to act via a
negative regulatory mechanism. Most likely, it represses the
transcription activity of one or more zinc finger transcription
factor(s) through a direct interaction. The next question is which
specific transcription factor(s) would be regulated by TA16. Until now,
only two zinc finger transcription factors, NGFI-A and Krox20, were
reported to be bound and repressed by NAB1 (30, 31). Many other zinc
finger transcriptional factors, including two members of the NGFI-A
family, NGFI-C and Erg3, were not affected by NAB1. This suggested that
NAB1 could only selectively regulate some specific transcriptional
activities. Since both NAGI-A and Brox20 transcription factors are able
to regulate several growth factors or proteins associated with cell
cycle progression (51, 52), they may be considered as candidate
regulatory proteins involved in the hormone-mediated cell growth arrest
if they are also expressed in these leiomyosarcoma cells. However,
other known or unknown transcription factors or transcriptional
activators with similar or different structural or functional features
of NGFI-A may also possibly be involved in this glucocorticoid-induced
cell growth suppression through interaction with TA16. Further, it
would also be interesting to know what kind of cell growth factor(s) or
cell cycle-related protein(s) are potentially regulated by the putative
transcription factor(s). In a recent study, we have demonstrated that
two growth-associated genes, Ha-ras and TGF-ß1, are down-regulated by
glucocorticoids in wild type DDT1 MF2 cells but not in GR1
cells (10). It is possible that these down-regulatory events are
mediated by TA16, particularly for the TGF-ß1, whose transcription
was known to be regulated by zinc finger transcription factor(s) (40).
Moreover, the glucocorticoid induction of TA16 may also possess other
biological functions. For example, we have recently demonstrated that
coadministration or pretreatment of DDT1 MF2 cells with
glucocorticoids could significantly inhibit apoptotic cell death
induced by taxol, an antimitotic agent (53). Further studies have
demonstrated that glucocorticoids inhibit taxol-induced apoptosis via a
mechanism independent of cell cycle arrest. Instead, it has suggested
that glucocorticoids may directly regulate one or more genes associated
with rescue of cells from apoptosis (53, 54). Thus, the discovery and
identification of TA16 as a transcription repressor have provided us
with a new approach to address these glucocorticoid-modulated
biological events.
In summary, we have described the identification and cloning of TA16, a
transcription repressor, by means of mRNA differential display and
other cloning procedures. The expression of this transcription factor
is markedly induced by glucocorticoids in wild type DDT1
MF2 cells, but only marginally changed in glucocorticoid-resistant
variant GR1 cells. Transfection assays demonstrated that introduction
of an antisense TA16 cDNA expression vector, controlled by a MMTV
promoter, into the DDT1 MF2 cell could significantly
relieve the glucocorticoid-induced cell growth arrest. In light of this
result and the promising biological functions of this class of
transcription repressors, it is possible that TA16 may play a critical
role in the signaling pathway underlying the glucocorticoid-induced
cell growth arrest in the leiomyosarcoma cells.
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MATERIALS AND METHODS
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Cell Culture and RNA Isolation
DDT1 MF2 cells and GR1 cells were cultured in DMEM
(GIBCO, Grand Island, NY) supplemented with 5% fetal FBS (Hyclone,
Logan, UT). Triamcinolone acetonide (TA) purchased from Sigma Chemical
Company (St. Louis, MO) was dissolved in 100% ethanol as
10-2 to 10-4 stock solution. TA was used at a
final concentration of 10-7 M in all
experiments unless otherwise noted. For RNA isolation,
10-7 M TA or 100% ethanol was added when the
cells were about 70% confluent. After 24 h of glucocorticoid
treatment, total RNA was isolated by the guanidine-thiocyanate
extraction method as previously described (55).
Messenger RNA Differential Display
Messenger RNA differential display was performed using RNAmap
kits A and B (GenHunter Corp., Brookline, MA) according to the
manufacturers instructions. Briefly, 0.2 µg of total RNA from
glucocorticoid-treated (10-7 M TA for 24
h) or untreated DDT1 MF2 cells was reverse transcribed,
respectively, with T12MG, T12MA,
T12MT, or T12MC (where M may be dG, dC, or dA)
as a primer, followed by PCR amplification in the presence of
[35S]dATP using the corresponding T12MN
(downstream) and one of the arbitrary 10 mers (AP110, upstream) as
primers. PCR-amplified fragments were loaded side by side on a 6%
sequencing gel. The dried gel was exposed to Kodak XAR film. The
reactions showing uniquely or overexpressed bands in
glucocorticoid-treated cells were repeated (reverse transcription and
PCR). The fragments reproducibly induced by glucocorticoids were
excised from the dried gels and reamplified by PCR using the
corresponding set of primers. The PCR fragments were then subcloned
into pCR II vectors (Invitrogen, San Diego, CA) according to the
manufacturers instructions. Subcloned cDNA fragments were then
sequenced in both direction using the Sequenase Version 2.0 DNA
sequencing kit (United States Biochemicals, Cleveland, OH). The novelty
of isolated clones was determined by a computer search and comparison
against Genbank and EMBL DNA databases.
Northern Blots
Total RNA isolated from glucocorticoid-treated or untreated
DDT1 MF2 and GR1 cells was fractionated in 1%
agarose-formaldehyde gels, transferred to Duralon filters
(Stragene), and UV cross-linked. Filters were probed with a
[32P]UTP-labeled antisense RNA probe generated from the
subcloned TA16 cDNA fragmented, which represents 286 bp of 3'-noncoding
region. Filters were then washed and autoradiographed as previously
described (33). The same filters were stripped and reprobed with a
hamster antisense ß-actin RNA probe to normalize for RNA loading.
Isolation of Intact cDNA of Clone TA16
A
ZAP cDNA library was constructed using mRNA isolated from
glucocorticoid-treated DDT1 MF2 cells (33). The subcloned
TA16 cDNA fragments, whose corresponding mRNAs have been confirmed by
Northern analysis as overexpressed in glucocorticoid-treated
DDT1MF2 cells, were used as probes for library screening.
Probe labeling and the procedure of library screening were performed as
previously described (32). The missing 5'-coding region was isolated by
the 5'-RACE System according to the manufacturers instructions (BRL,
Gaithersburg, MD). The following three antisense primers were
synthesized according to the cDNA sequence of Clone SK-TA16-A2:
5'-TCAGTCTGCTGTGAACTCAGG-3', 5'-CTCCTCACTCTTGGCTCTGG-3', and
5'-AGGTTCCTGGAAGTCTGG-3'. Total RNA isolated from the DDT1
MF2 cells treated with glucocorticoid (10-7 M
TA) for 24 h was used for the 5'-RACE PCR (34). The PCR products
were subcloned into pCR II vectors and sequenced. Finally, utilizing
the unique XhoI restriction site in the TA16 cDNA, two
separate pieces of cDNAs were joined by ligation and inserted into pCR
II vector (PCR II-TA16, see Fig. 2
).
Construction of Expression Vectors
Sense and antisense TA16 expression vectors were constructed by
utilizing the unique restriction sites available with the pCR II vector
and the MAMneo-MMTV vector. As shown in Fig. 5
, for construction of a
sense TA16 vector, full-length cDNA sequence was cut from the
pCRII-TA16 vector and inserted in the pMAMneo-MMTV expression vector.
For construction of an antisense vector, only part of the TA16 cDNA
(from nucleotide 1 to 968) was cut out from the pCRII vector and
religated into MAMneo-MMTV vector. All constructed expression vectors
were confirmed by DNA sequencing.
Stable Transfection and Selection of Transfectant Cell Lines
Transfections were performed by lipofectin (BRL) as recommended
by the manufacturer. Briefly, the cells were washed twice with Opti-MEM
reduced serum medium, and 3 ml of the same reduced-serum medium were
added to the cells. Plasmid DNA (2 µg per 6-cm plate) containing
either sense or antisense TA16 inserts was mixed with lipofectin before
they were added to the tumor cells. After transfection, stable
transfectants were selected by incubating the cells in the medium
containing 500 µg Genecitin (G418)/ml. After approximately 2 weeks,
the cells without transfection of expression vectors were dead. To
select the single colonies of transfectants, diluted cells were
transferred into 96-well plats with a calculated density at 0.7 cell
per well. After 1215 days, a number of wells exhibited colonies that
had proliferated from a single cell. These colonies of cells were then
collected and amplified for further experiments.
Determination of Cell Growth
As previously described (10), wild type DDT1 MF2
cells or the cells transfected with TA16 expression vectors were
cultured in 24-well plates at an initial density of 104
cells per well. Cells were then treated with 10-7
M TA or 100% ethanol (1 µl/ml), respectively. Cell
growth was monitored daily using a Coulter Counter. After 12 days of
cell culture, cells were observed and photographed using phase contrast
microscopy.
RNase Protection Assays
[32P]UTP-labeled RNA transcripts (189 bp)
complementary to antisense TA16 mRNA sequence were synthesized by Ambio
T3 MAXIscript kit (Amobion, Austin, TX) from SK-TA16-A2 vector digested
with XhoI (see Fig. 2
). Total RNA from untransfected
DDT1 MF2 cells or the cells transfected with antisense TA16
expression vectors was hybridized overnight at 42 C with the RNA probe.
The remaining steps for the RNase protection was performed by using the
RPA II kit (Ambion, Austin TX) according to the manufacturers
instructions. The samples were then analyzed on a 6% denaturing
acrylamide gel, followed by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Mark Willingham and Merrill Miller for their
critical comments on this manuscript. We also express our appreciation
to Qiaoliang Fang and Chvonne Simmons for their excellent technical
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Weimin Fan, M.D. Department of Pathology and Laboratory Medicine, 171 Ashley Avenue, Charleston, South Carolina 29425.
This work was supported by NIH Grant CA-58846 (to W.F.) and the Health
Science Foundation of the Medical University of South Carolina.
Received for publication March 18, 1997.
Revision received April 23, 1997.
Accepted for publication May 6, 1997.
 |
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