Molecular Cloning and Biological Activity of a Novel Ha-Ras Suppressor Gene Predominantly Expressed in Skeletal Muscle, Heart, Brain, and Bone Marrow by Differential Display Using Clonal Mouse EC Cells, ATDC5*

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.

The mammalian ras protooncogenes, Ha-, Ki-, and N-ras, are expressed in a variety of tissues (1). For example, Ha-ras is highly expressed in skin and skeletal muscle; Ki-ras in gut and thymus; and N-ras in testis and thymus. Ras proteins bind guanine nucleotides and possess intrinsic GTPase activity, serving as transducers of diverse physiological signals including those controlling cellular proliferation and differentiation (2). Some of the biological activities of Ras proteins are known to be modulated by other proteins, including Krev-1 (3) and Ha-rev107 (4). However, physiological functions of Ras proteins and these regulatory proteins in mammalian cells remain largely unknown.
Chondrogenesis is a key event in skeletal development in vertebrates. We previously reported that chondrogenesis could be induced in chondroprogenitor-like EC cells, ATDC5, at a high incidence when cultured in the presence of insulin and that ATDC5 cells keep track of the overt chondrogenesis in vitro (5)(6)(7)(8)(9). Clonal mouse embryonic fibroblast cells, C3H10T1/2, retain the properties of pluripotent mesodermal progenitors and have been shown to differentiate into adipocytes, myoblasts, osteoblasts, as well as chondrocytes (10) under distinct cultural conditions, including the presence of 5-azacytidine or high dose bone morphogenetic protein-2 (10,11). In this study, comparison by differential display of mRNAs expressed in undifferentiated ATDC5 cells with those in undifferentiated C3H10T1/2 cells led us to isolate a novel cDNA clone encoding a Ha-rev107-related protein predominantly expressed in skeletal muscle, heart, hippocampus, and bone marrow as well as ATDC5 cells.

EXPERIMENTAL PROCEDURES
Cells and Culture Conditions-ATDC5 cells were plated in six-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cultured as described previously (5,6). Clonal mouse embryonic fibroblast C3H10T1/2 cells (10), clonal mouse fibroblast NIH3T3 cells (13), clonal mouse fibroblast Balb/c 3T3 cells (14), and clonal mouse myoblast C2C12 cells (15) (RIKEN Cell Bank, Tsukuba, Japan) were plated in six-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cultured for 3 days in DMEM 1 containing 10% fetal bovine serum (FBS). Clonal mouse newborn calvaria-derived osteogenic MC3T3-E1 cells (16) and clonal mouse stromal ST2 cells (17) were plated in six-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cultured for 3 days in ␣ minimal essential medium containing 10% FBS. Cultured adherent cells from bone marrow were prepared from 6-week-old ICR mice (SRL, Hamamatsu, Japan) as described previously (18) with some modifications. Briefly, tibiae and femurs were dissected, the ends of the bones were cut, and bone morrow was flushed out with DMEM/Ham's F-12 hybrid medium containing 10% FBS. The pooled marrow cells were dispersed by agitation in the syringe and plated in six-multiwell plastic culture plates (2 ϫ 10 7 cells/well). After 48 h, nonadherent cells were removed by replacing the medium and the adherent cells were cultured further for five days at 37°C in a humidified 5% CO 2 , 95% air atmosphere with medium replacement every other day.
RNA Extraction and Differential Display-Total RNA was isolated from C3H10T1/2 cells and ATDC5 cells by a single-step method as described previously (6) and analyzed by differential display according to the manufacture's instruction (RNAmap TM , GenHunter, Nashville, TN). The DNA fragment of approximately 500-bp expressed only in undifferentiated ATDC5 cells was identified and subcloned into pCRII vector (Invitrogen, San Diego, CA), and its nucleotide sequence was * This work was supported by a grant from the Japan Orthopaedics and Traumatology Foundation, Inc. (No. 0094). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  cDNA Library Construction and Isolation of A-C1 cDNA-Oligo(dT)primed cDNA library from undifferentiated ATDC5 poly(A) ϩ RNA was constructed in ZAP Express vector (Stratagene, La Jolla, CA), and 1 ϫ 10 6 plaques were screened with the 500-bp fragment as a probe. Plaques were transferred to the membranes (137-mm nylon membrane, NEN Life Science Products), the PCR fragment was 32 P-labeled (BcaBEST labeling kit, Takara, Otsu, Japan), and hybridization was performed in 6 ϫ SSPE, 0.2% bovine serum albumin, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, 0.1% SDS, 100 g/ml denatured salmon sperm DNA, and the 32 P-labeled probe for 16 h at 42°C. The membranes were washed to a final stringency of 0.1 ϫ SSPE (3 M NaCl, 197 mM, NaH 2 PO 4 , 25 mM EDTA) and 0.1% SDS at 55°C.
Northern Analysis-ATDC5 cells and C3H10T1/2 cells were plated in six-multiwell plastic plates and cultured as described above. Total RNA was isolated and analyzed by Northern hybridization as described previously (6). Briefly, poly(A) ϩ RNA (5 g) was denatured, separated by 1% agarose gel electrophoresis, and transferred on Nytran membranes (Schleicher & Schuell, Dassel, Germany). A 3.0-kb cDNA fragment of A-C1 was used for hybridization as a probe. In analysis of tissue distribution in adult mice, a labeled cDNA was hybridized to a mouse multiple tissue Northern blot (CLONTECH, Palo Alto, CA). After hybridization, the membranes were exposed to X-Omat films (Eastman Kodak Co.) at Ϫ80°C with Cronex lightening plus intensifying screens (DuPont).
RT-PCR-The RT-PCR was performed as described previously (6). Briefly, first-strand cDNA was synthesized using SuperScript II RNase H-reverse transcriptase (Life Technologies, Inc.) with 5 g of total RNA extracted from various cell lines cultured in vitro. The following specific primers were used: 5Ј-CACACTGGTAAGTGGGGCAAGACCG-3Ј (sense primer) and 5Ј-GGATTGTGTTGTTTCAGGGTTCGGG-3Ј (antisense primer) for mouse A-C1 cDNA. Amplification consisted of initial denaturation at 94°C for 5 min, followed by 25 reaction cycles (30 s at 94°C, 30 s at 60°C, and 30 s at 72°C). Aliquots (8 l) of each PCR products were resolved on 3% NuSieve 3:1 agarose gels (FMC BioProducts, Rockland, ME) alongside the markers. The amplified PCR products were subcloned into pCRII vector (Invitrogen, SanDiego, CA), and the nucleotide sequences of all cDNA fragments were verified using ALFred DNA Sequencer (Amersham Pharmacia Biotech).
In Situ Hybridization-Brains and tibiae of male 5-week-old ICR mice were collected and fixed in 4% paraformaldehyde in 10 mM phosphate-buffered saline (pH 7.4) overnight at 4°C. Tibiae were decalcified for 4 days in 10% EDTA. They were dehydrated in a graded series of ethanol and embedding in paraffin. Sections (6 m thick) were then processed for in situ hybridization as described previously (19). The 3.0-kb mouse A-C1 cDNA was labeled with [ 35 S]TTP (DuPont Biotechnology Systems, Boston, MA) by nick translation to a specific activity of 3.0 -5.0 ϫ 10 8 cpm/g. After hybridization, the slides were washed under conditions of high stringency, and the dried tissue sections were dipped into NTB-2 emulsion (Kodak) and exposed for 7 days at 4°C. The sections were counterstained with hematoxylin. Specificity of this cDNA probe was confirmed by Northern blot analysis with total RNA extracted from ATDC5 cells (Fig. 5). In addition, the sections pretreated with RNase before in situ hybridization with the cDNA probe showed no autographic signals, indicating that the hybridization signals were dependent on the presence of RNA (data not shown).
In Vitro Transcription/Translation-A coupled transcription/translation reaction was performed using the rabbit reticulocyte lysate system (TnT Coupled Transcription/Translation Systems, Promega, Madison, WI) in the presence of [ 35 S]methionine (Amersham Pharmacia Biotech, catalog number AG1094) according to the manufacturer's instructions. The 3.0-kb A-C1 cDNA cloned into pcDNA3.1 vector (Invitrogen, San Diego, CA) was used as a template (pCMV-AC1). The translation product was electrophoresed on a 10 -20% polyacrylamide gel and detected by autoradiography with the X-Omat films (Kodak) (8-h exposure at room temperature). Prestained rainbow marker (Amersham Pharmacia Biotech) was loaded in the adjacent lane to estimate molecular sizes.
Expression and Detection of FLAG-tagged A-C1 Protein-A DNA fragment encoding the A-C1 protein appended with a FLAG tag at its C terminus was generated by PCR using the 3.0-kb A-C1 cDNA as a template and the primer sequences as follows: sense primer 5Ј-GCCG-CCACCATGGACCCGACACGGTCCC-3Ј; antisense primer 5Ј-CTACT-TGTCATCGTCGTCCTTGTAATCATATTTCGTTCTTTGTCTTTTGGG-AAAC-3Ј. The antisense primer contained sequences for a FLAG tag (underlined) and a stop codon. Amplification consisted of initial denaturation at 94°C for 5 min, followed by 25 reaction cycles (30 s at 94°C, 30 s at 60°C, and 30 s at 72°C). The PCR product was gel-purified and cloned into pCRII vector (pCRII/A-C1FLAG), and its nucleotide sequence was verified by ALFred DNA Sequencer. The insert of the pCRII/A-C1FLAG vector was excised with SpeI and XhoI and recloned into the SpeI-XhoI site of pcDNA3.1 vector. The resultant expression vector, pCMV/A-C1FLAG, or pcDNA3.1 as a control were transiently transfected into COS-7 cells cultured on Lab-Tec Chamber Slide (Nalge Nunc International, Naperville, IL) by lipofection using SuperFect Transfection Reagent (Qiagen, GmbH, Hilden, Germany). Two days later, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.01% Triton X-100, and then incubated with diluted normal blocking serum for 20 min at room temperature. Incubation of these cells with anti-FLAG M2 monoclonal antibody was performed for 30 min at room temperature. The bound antibody was detected using the Vectastain ABC Kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). After three rinses with phosphate-buffered saline, staining was developed for 10 min using the Dako AEC Substrate System (Dako Corp.). Slides were mounted with Crystal Mount (Biomeda, Foster City, CA) and examined under a microscope.
Transfection of A-C 1 cDNA into Ha-ras-and Ki-ras-transformed NIH3T3 Cells-Lras/NIH cells are a derivative of NIH3T3 containing human Ha-ras 12V oncogene, 2 and DT is a derivative of NIH3T3 (HG-PRT Ϫ ) containing two copies of Kirsten murine sarcoma virus provirus (20). Lras/NIH cells and DT cells were cultured in DMEM supplemented with 10% FBS, 0.03% L-glutamine, 100 g/ml penicillin G, and 100 g/ml streptomycin sulfate (growth medium). The transfection protocol we used was a modification of the method described previously (3). pCMV-AC1, pcDNA3.1, or pKrev-1 (a Krev-1 expression vector, positive control) (2.5 g), pSV-BSD (blasticidin-S-resistant vector) (1.5 g), and sheared calf thymus DNA (1 g) as a carrier were used. Lras/NIH cells and DT cells (3 ϫ 10 4 ) were plated 1 day prior to co-transfection on a collagen-coated 35-mm diameter dish. The DNA-CaPO 4 co-precipitates were formed in 100 l volume and added to the cells covered with 1.5 ml growth medium according to the method of Wigler et al. (21). After incubation at 37°C for 12-16 h in a CO 2 incubator, the cells were treated with 1 ml of 25% glycerol in 1 ϫ DMEM for 1 min, rinsed with DMEM, and refed with 2 ml of growth medium. On the following day, the cells were trypsinized and replated onto a 100-mm diameter dish with 15 ml of growth medium supplemented with 8 g/ml blasticidin-S (Funakoshi, Tokyo, Japan). The medium was changed on the next day and 4 days later. After 4 days of selection with blasticidin-S, total number of colonies were counted under a phase-contrast microscope. The number of the flat clones was counted on the following day. To determine the growth curve, Lras/NIH cells transfected with pCMV-AC1 or pcDNA3.1 vector were inoculated in 24-multiwell plates and cultured with the growth medium containing 8 g/ml blasticidin-S. The cells were rinsed with phosphate-buffered saline twice, treated with trypsin and counted with a hemocytometer at the time points indicated.

RESULTS
Cloning and the Structure of A-C1 cDNA-To find genes specifically expressed in chondrogenic cells, we performed differential display using the total RNA extracted from a chondrogenic EC cell line, ATDC5, and obtained a 500-bp cDNA fragment corresponding to the 3Ј-untranslated sequence of a novel differentially expressed gene named A-C1. This cDNA fragment was then used as a probe to screen at high stringency a mouse cDNA library generated from ATDC5 cells. Ten cDNA clones were obtained from 1 ϫ 10 6 independent plaques. Eight out of ten clones contained a cDNA of about 3.0 kb. Fig. 1A shows the nucleotide sequence of A-C1 cDNA and the deduced amino acid sequence of the putative A-C1 protein. A-C1 cDNA contains a 501-bp open reading frame starting from an ATG codon. The sequence surrounding this ATG well matches the Kozak consensus sequence (22). Therefore, A-C1 protein is predicted to consist of 167 amino acid residues. The predicted A-C1 protein has a calculated molecular mass of 18,809 daltons and an isoelectric point of 6.1. The 3Ј-end of the sequence contains a poly(A) stretch, preceded by a putative polyadenylation signal (AATAAA). A hydrophobicity plot using the Kyte-Doolittle algorithms (23) showed the existence of a possible transmembrane domain at its C ternimus (143-159 amino acid) (Fig. 1B). The amino acid sequence lacks the N-terminal signal peptide. These data suggest that the A-C1 cDNA is likely to encode an intracellular, membrane-bound protein. The amino acid sequence of A-C1 protein showed a significant homology with that of rat Ha-rev107 (4) (46% amino acid identity at the amino acid level) (Fig. 2).
Expression of A-C1 Protein by in Vitro Transcription/Translation-To confirm that the cloned 3.0-kb A-C1 cDNA contained a functional open reading frame, we performed an in vitro transcription/translation using the rabbit reticulocyte lysate system in the presence of [ 35 S]methionine. A single protein product of the expected size was detectable. (Fig. 3). No protein product was detectable with the vector control (pcDNA3.1) (data not shown).

Transient Expression and Subcellular Localization of A-C1
Protein-To analyze the subcellular localization of A-C1 protein (Fig. 4), COS-7 cells were transiently transfected with a pcDNA3.1 plasmid containing the A-C1 cDNA with a FLAG tag sequence at the C terminus (pCMV/A-C1FLAG). Immunocytochemical staining with monoclonal anti-FLAG M2 antibody showed that A-C1 protein was localized in the cytoplasm and the perinuclear region but not within the nucleus. No staining was seen after transient transfection of the vacant pcDNA3.1 vector.
Expression of A-C1 mRNA in Various Cell Lines-We assessed by Northern analysis and RT-PCR the expression of A-C1 mRNA in culture cell lines: ATDC5, C3H10T1/2, MC3T3-E1, NIH3T3, Balb/c 3T3, ST2, and C2C12. A major hybridization band of about 3.2 kb was detected in chondrogenic ATDC5 cells, but not in C3H10T1/2 cells (Fig. 5A). The expression of A-C1 mRNA was detectable by RT-PCR in C2C12 as well as ATDC5, but not in MC3T3-E1, NIH3T3, Balb/c 3T3, and ST2 (Fig. 5B).
Expression of A-C1 mRNA in the Adult Mice Tissues-Northern analysis showed that among the various adult mice tissues, A-C1 mRNA was expressed highly in skeletal muscle and moderately in heart and brain (Fig. 6A). The expression of A-C1 mRNA was detectable also in bone marrow cells by RT-PCR (Fig. 6B). By in situ hybridization, A-C1 mRNA was localized in hippocampus and bone marrow in adult mice (Fig. 7).
Biological Activity of A-C1-The deduced amino acid sequence of A-C1 showed homology with that of rat Ha-rev107, suggesting that A-C1 may possess the revertant-inducing activity on ras-transformed NIH3T3 cells. We assessed such activity by transfecting A-C1 gene into NIH3T3 cell line transformed by Ha-ras 12V or v-Ki-ras oncogene (Lras/NIH and DT, respectively) and observing the morphology. When Lras/NIH cells were transfected with pCMV-AC1, the colonies of these transfectants, observed under a phase-contrast microscope, were relatively smaller in size than those in the control culture transfected with pcDNA3.1 vector. Moreover, some of these

FIG. 3. Translation of A-C1 gene in vitro.
A coupled transcription/ translation reaction (TnT Reticulocytes, Promega) was performed using A-C1 cDNA cloned into pcDNA3.1 vector as a template. The translation products were separated by electrophoresis on a gradient polyacrylamide gel (10 -20%) and detected by autoradiography. Three independent experiments were performed and gave similar results. colonies consisted of flat cells with increased attachment to the substrate (Fig. 8). The frequency of flat colonies was comparable with that observed after transfection of the control Krev-1 gene (Table I). Total numbers of colonies, however, were similar between the vector-transfected and pCMV-AC1-transfected cultures. In contrast, pCMV-AC1 did not give rise to flat colonies when transfected into DT cells (Fig. 8), while Krev-1 showed a substantial activity to induce flat colonies in this cell line ( Table I).
Overexpression of A-C1 suppressed not only the transformed morphology of Lras/NIH cells but also the growth of these cells (Fig. 9). Doubling times of the pooled A-C1-transfected and vector-transfected Lras/NIH cells were ϳ20 and ϳ34 h, respectively. Saturation densities, however, were similar between the two (ϳ2.2 ϫ 10 5 cells/cm 2 ). DISCUSSION We have isolated by differential display a novel gene, A-C1, encoding a protein of 167 amino acids, which is specifically expressed in skeletal muscles, heart, brain, and bone marrow A DNA fragment of ␤-actin was also hybridized to the same blots as a control. B, total RNA was isolated from the cultured adherent bone marrow cells. RT-PCR was performed, and aliquots (8 l) of the PCR product were resolved on 3% agarose gels. Three independent experiments were performed and gave similar results.
in vivo. A-C1 protein has a putative transmembrane domain at the C ternimus (143-159 amino acid) and lacks the N-terminal signal peptide (Fig. 1), indicating that A-C1 protein is probably an intracellular, membrane-bound protein. Indeed, a preliminary experiment with tagged protein described here suggested its predominant localization in the cytoplasm and perinuclear region (Fig. 4). Further studies are required, however, to confirm the subcellular localization and membrane-association of the intact, endogenous A-C1 protein under physiological conditions.
The amino acid sequence of A-C1 showed 46% homology with that of rat Ha-rev107 (4). Ha-rev107 is a class II tumor suppressor, as defined by its down-regulation after Ha-ras transformation in fibroblasts, expression in ras-resistant fibroblasts, and growth-inhibiting capacity in Ha-ras-transformed cell lines. The expression of Ha-rev107 was detected in liver, kidney, stomach, and intestine, distinct from that of A-C1, while Ha-rev107 protein appeared to be linked to the nuclear membrane and to membranes in the perinuclear space, similar to the subcellular localization of A-C1. Ras proteins bind guanine FIG. 7. Localization of A-C1 gene expression in brain and bone marrow in adult mice. Brains and tibiae of 5-week-old mice were fixed, dehydrated, and embedded in paraffin. Sections (6 m thick) were processed for in situ hybridization as described under "Experimental Procedures." Silver grains were accumulated in hippocampus in brain and bone marrow in diaphysis of tibiae. Three independent experiments were performed and gave similar results.   nucleotides with high affinity. Three sequence motifs important for nucleotide interaction have been determined, which are conserved between different guanosine nucleotide-binding proteins (12): GXXGXGKS is involved in the binding to the ␣and ␤-phosphates; DXXG is involved in binding to Mg 2ϩ and ␥-phosphate when GTP is bound; and NKXD is important for binding to the guanine ring. Both A-C 1 and Ha-rev107 protein have two consensus sequence motifs, DXXG and NKXD (Fig. 2), which were previously unnoticed. The role of these motifs should be elucidated in future studies. Overexpression of A-C1 not only induced reversion of morphology in Ha-ras-transformed NIH3T3 cells but also suppressed the growth of these cells, as evidenced by the smaller colony formed (Fig. 8) and the growth curve (Fig. 9). These results support the notion that A-C1 may serve as a negative regulator for the Ha-ras-mediated signaling pathway. How this is achieved is also an important subject for future studies. Our data also indicated that A-C1 is not an effective suppressor against the v-Ki-ras-transformed cell line we used. Whether this reflects some kind of selectivity among Ras family proteins is an interesting question to be clarified.
In this study, we identified the A-C1 gene by differential display to screen genes specifically expressed in mouse chondrogenic EC cells, ATDC5. Our observations that A-C1 is expressed in specific tissues in vivo and certain cell lines in vitro and that A-C1 is a potential inhibitor of Ha-ras-mediated intracellular signaling pathway raise the possibility that A-C1 may play a role in the regulation of cell growth and differentiation in these specific tissues.