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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (AP1–10) 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. 1Go).



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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.

 
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. 2Go, 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. 2Go). 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. 3Go). 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. 4Go), 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.

 
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. 5Go). 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. 6Go, 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. 6Go). 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. 7Go). 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. 8Go, 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. 6Go. 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 8–10 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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. 1Go). 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. 6Go and 7Go). 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. 6Go). 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-{kappa}B by its sequestration in the cytoplasm by I{kappa}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 (AP1–10, 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 manufacturer’s 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 {lambda}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 manufacturer’s 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. 2Go).

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. 5Go, 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 12–15 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. 2Go). 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 manufacturer’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Norris JS, Gorski J, Kohlor PO 1974 Androgen receptor in a Syrian hamster ductus deferens tumor cell line. Nature 284:422–424
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