Overexpression of Bamacan/SMC3 Causes Transformation*

Bamacan can occur in certain cell types as either a secreted proteoglycan assembled into basement membranes or as an intracellular protein known as structural maintenance of chromosome 3 (SMC3). To assess the role of this protein in tumorigenesis, we investigated whether induced overexpression of bamacan/SMC3 could transform normal fibroblasts. We generated a full-length cDNA encoding the entire mouse bamacan/SMC3 and demonstrated appropriate transcription and translation into a 146-kDa protein. All the NIH and Balb/c 3T3 murine fibroblasts overexpressing this bamacan/SMC3 transgene generated foci of transformation and acquired anchorage-independent growth. The increased levels of bamacan/SMC3 expression achieved in the transfected fibroblasts were the same as those detected in a series of spontaneously transformed murine and human colon carcinoma cells. Moreover, a 3-4-fold overexpression of bamacan/SMC3 was detected in ∼70% of human colon carcinoma specimens from matched pairs (n = 19, p < 0.0002) and in a cohort of intestinal tumors fromApc-deficient Min/+ mice. These results support the concept that deregulated expression of bamacan/SMC3 is involved in cell transformation.

Bamacan can occur in certain cell types as either a secreted proteoglycan assembled into basement membranes or as an intracellular protein known as structural maintenance of chromosome 3 (SMC3). To assess the role of this protein in tumorigenesis, we investigated whether induced overexpression of bamacan/SMC3 could transform normal fibroblasts. We generated a fulllength cDNA encoding the entire mouse bamacan/SMC3 and demonstrated appropriate transcription and translation into a 146-kDa protein. All the NIH and Balb/c 3T3 murine fibroblasts overexpressing this bamacan/SMC3 transgene generated foci of transformation and acquired anchorage-independent growth. The increased levels of bamacan/SMC3 expression achieved in the transfected fibroblasts were the same as those detected in a series of spontaneously transformed murine and human colon carcinoma cells. Moreover, a 3-4-fold overexpression of bamacan/SMC3 was detected in ϳ70% of human colon carcinoma specimens from matched pairs (n ‫؍‬ 19, p < 0.0002) and in a cohort of intestinal tumors from Apc-deficient Min/؉ mice. These results support the concept that deregulated expression of bamacan/ SMC3 is involved in cell transformation.
Originally isolated from the embryonic parietal yolk sac as a high density chondroitin sulfate proteoglycan (1,2), bamacan was subsequently identified to be a component of the renal mesangial matrix (3), the basement membrane of other tissues (4), and tumor matrix (5). The murine protein is encoded by 31 exons distributed along ϳ45 kilobase pairs of genomic DNA (6) and is highly conserved across species (7)(8)(9). Secondary structure analysis of the protein reveals three globular domains intercalated by two ␣-helix coiled-coils in an antiparallel arrangement folded at a flexible hinge (10). The terminal ends harbor five glycanation sites to which the attachment of glycosaminoglycan side GAG chains is possible, and a P-loop and a DA-box motif that may act cooperatively to bind ATP and DNA, respectively (11). These features and the high sequence homol-ogy with the structural maintenance of chromosome 3 (SMC3) 1 protein led us to conclude that bamacan is a member of this family (6). SMC3 proteins are involved in the formation of at least two multimeric complexes within the nucleus. The first, termed "cohesin", is involved in chromosome cohesion and prevents premature sister chromatid separation (12). The second, called RC-1, is involved in DNA repair and has DNA ligase and polymerase activity (13). The formation of a third complex has been postulated on the basis that bamacan/SMC3 can be coisolated with proteins involved in microtubule dynamics (9). Finally, the protein has a functional leucine zipper domain that binds Mxi and Max, two nuclear proteins that interact with Myc and modulate its transcriptional activity (14).
We previously detected increased bamacan/SMC3 mRNA levels in several transformed cell lines (6), suggesting that the amplified product of the normal gene may play a role in tumorigenesis. To directly test this hypothesis we investigated the effects of transgenic overexpression of bamacan/SMC3 in NIH and Balb/c 3T3 mouse fibroblasts. All the cells overexpressing bamacan/SMC3 showed evidence of transformation, including anchorage-independent growth and foci of transformation. Moreover, all the stably transfected clones exhibited increased levels of bamacan/SMC3 identical to those of spontaneously transformed human colon carcinoma cell lines and colon cancer specimens, as well as intestinal tumors from Apc-deficient Min/ϩ mice. Thus, deregulated expression of bamacan/SMC3 may be directly linked to the complex process of oncogenesis.

EXPERIMENTAL PROCEDURES
Materials and Cells-[␣-32 P]dATP (ϳ3000 Ci/mmol) and [ 35 S]methionine (ϳ1000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. The transcription/translation TNT T7 kit was from Promega. Rabbit anti-mouse bamacan/SMC3 antibody was kindly provided by A. Strunnikov. Cell lines include: NIH and Balb/c 3T3 mouse fibroblasts that display contact inhibition and serum dependence; a non-tumorigenic colon epithelial cell line originated from p53 Ϫ/Ϫ mice (15); CMT-93 mouse colorectal carcinoma cells; and the human colon carcinoma cell lines HCT116, WiDr, SW480, LOVO, and CaCo2 (American Type Culture Collection). Nineteen human colon carcinoma specimens and matched normal tissues, and five Min/ϩ animals were also investigated.
Generation of Full-length Bamacan cDNA and Expression Vectors-Full-length bamacan cDNA was assembled by sequential ligation of EST clones AA144255, AA164123, and AA119169 and of a cDNA fragment generated by reverse transcriptase polymerase chain reaction (RT-PCR). Because of the lack of suitable subcloning vectors, one was generated from pBS-KSϩ by replacement of the multiple cloning site between the KpnI and the NotI with a 24-bp oligonucleotide corresponding to the restriction sites KpnI, SpeI, XhoI, NcoI, SalI, NotI (5Ј to 3Ј). These new sites allowed the sequential subcloning of the various cDNAs. The coding region of bamacan/SMC3 was inserted at the NotI/ EcoRV sites of the pcDNA3.1 myc-His (Invitrogen). A pLXSN retroviral expression vector (CLONTECH) was also constructed. All ligations were verified by restriction map digestion and DNA sequencing.
Stable Transfection, Tumorigenic Assays, and Northern and Western Blotting-Balb/c 3T3 cells were transfected with 50 g of expression vector by electroporation (230 V, 1080 microfarads/s). After 2 days, G418 (250 g/ml) was added and drug selection carried out for 2 weeks. Single colonies were ring-cloned and expanded. Ten days later, RNA was extracted from a first group of cells whereas the parallel group was * This work was supported by National Institutes of Health Grants RO1 CA39481 and RO1 CA47282 (to R. V. I). 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.
¶ To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Rm. 249, JAH, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail: iozzo@lac.jci. tju.edu. fixed in 70% ethanol, stained with 0.2% methylene blue, and analyzed for the presence of foci of transformation. Growth in soft agar and colony formation was determined by culturing 3 ϫ 10 3 cells between two layers of 0.4% (bottom) and 0.2% (top) agar solution. Colony size was scored at 14 and 21 days by counting the number of cell aggregates Ͼ100 m diameter in several randomly selected 9-mm 2 areas. Bamacan mRNA levels were assessed by Northern blotting as described previously (6). Cellular levels of bamacan/SMC3 were assessed by Western immunoblotting. Briefly, the cells were solubilized in phosphate-buffered saline containing 0.1% Triton X-100 and a mixture of protease inhibitors Complete TM (Roche Molecular Biochemicals), separated by SDS-PAGE, and immunoblotted using the primary antibody at 1:300 dilution and the secondary (peroxidase-conjugated anti-rabbit IgG) at 1:17,000 dilution, followed by chemiluminescence (Pierce) detection.

Generation of an Expression Vector Encoding the Full-length
Bamacan/SMC3 cDNA and in Vitro Transcription/Translation-To assess the role of bamacan in tumorigenesis, we first needed to construct a full-length eukaryotic expression vector. The strategy for the assembly of full-length mouse bamacan/ SMC3 cDNA, and the pcDNA3.1 and retroviral pLXSN expression vectors, is illustrated in Fig. 1, A and B. The full-length cDNA encompasses 3672 bp and encodes 1206 amino acid residues, including a C-terminal stretch of 11 residues that are not present in the rat sequence (7). To assess whether the predicted open reading frame of the bamacan/SMC3 cDNA could be translated into a protein, the pcDNA3.1 vector was tested in a rabbit reticulocyte lysate transcription-translation system using [ 35 S]methionine as the precursor. The results showed a single protein of the expected size of ϳ146 kDa (Fig. 1C, lanes  1 and 3). Lack of the expression vector in the reaction mixture generated no detectable protein (Fig. 1C, lane 2), whereas addition of the vector encoding luciferase gave the predicted band of ϳ60 kDa (Fig. 1C, lane 4). Thus, the full-length cDNA encoding bamacan protein is appropriately transcribed and translated into a protein of size (146 kDa) identical to the native bamacan/SMC3.
Overexpression of Bamacan/SMC3 Causes Transformation of Normal Murine Cells-Because bamacan mRNA levels are elevated in a number of tumorigenic cell lines (6), we investigated whether overexpression of this protein could directly produce cell transformation. For this purpose, normal NIH and Balb/c 3T3 fibroblasts, which are both contact inhibited, were transfected with the pcDNA3.1 bamacan/SMC3 expression vector. Of the 22 clones obtained, 3 displayed ϳ4-fold bamacan overexpression ( Fig. 2A). Eighteen of these clones were tested for the ability to generate colonies in soft agar. In these experiments, clones 9 and 22, i.e. those overexpressing bamacan mRNA, displayed anchorage-independent growth and generated 31 Ϯ 4 (n ϭ 3) and 23 Ϯ 4 (n ϭ 4) colonies Ͼ100 m diameter after 3 weeks of growth (Fig. 2B). No colonies were observed in the remaining 16 G418-resistant clones expressing normal bamacan mRNA and in wild type cells. Growth kinetics experiments revealed that the overexpressing clones had lost contact inhibition, confirming they had acquired a transformed phenotype. Initial growth rate did not differ significantly between overexpressing and normally expressing bamacan clones. However, upon reaching confluence, normally expressing clones significantly reduced their duplication rate to ϳ0.2 doubling/day, whereas clones 9 and 22 maintained a vigorous duplication activity (ϳ0.6 -1 doubling/day) (Fig. 2C). All the G418-resistant clones were tested in a transformation focus assay. Whereas wild type and G418-resistant clones expressing normal bamacan/SMC3 mRNA levels did not generate foci of transformation, clones 9 and 22 generated numerous foci of transformation (Fig. 2D). Moreover, cells expressing normal bamacan level (clone 6) formed a uniform monolayer, whereas cells overexpressing bamacan exhibited an haphazard arrangement and lacked contact inhibition (Fig. 2E). In agreement with the Northern blotting data, the cellular levels of bamacan/ SMC3 protein in the overexpressing clones were ϳ3-fold higher than control (Fig. 2F). In contrast, no bamacan levels could be detected in the media conditioned by either wild type or transfected cells (not shown).
To corroborate the transformation potential of bamacan, we performed additional experiments in which bamacan/SMC3 gene was delivered into NIH 3T3 cells using pLXSN retroviral vector. Among 12 clones isolated, one displayed increased bamacan/SMC3 mRNA (clone 1, Fig. 3A) and protein (Fig. 3B) levels. As in the cells transfected with the pcDNA3.1 vector, the retroviral clones overexpressing bamacan formed numerous foci of transformation (Fig. 3C).
Collectively, these findings support the conclusion that induction of intracellular bamacan/SMC3 expression is sufficient to cause transformation of normal, contact-inhibited fibroblasts.
Bamacan Expression Is Abnormally Elevated in Colon Carcinoma Cell Lines and Colon Carcinoma Tissues-To assess whether the degree of bamacan/SMC3 expression that caused transformation of 3T3 fibroblast was similar to that of established neoplastic cells, a series of mouse and human neoplastic cell lines was examined. A non-tumorigenic colon epithelial cell line originated from p53 Ϫ/Ϫ mice was used as control. These cells do not grow in soft agar and do not generate tumors in either SCID or immunocompetent syngeneic mice (15). Compared with the expression in this primary colon cell line, a 5-fold increase in bamacan/SMC3 expression was observed in-CMT-93 mouse colorectal carcinoma cells (Fig. 4A). Similar

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results were obtained when normal colon tissue and the matching neoplastic tissue from a patient with colon carcinoma were investigated (Fig. 4B). Five independent human colon carcinoma cell lines displayed the same degree of bamacan overexpression as the colon carcinoma sample (Fig. 4B), suggesting that the phenomenon does not simply reflect the high rate of mitosis of the cells held in culture. We then compared bamacan/ SMC3 expression in matched neoplastic and normal colon tis-sues (n ϭ 19). About 70% of the colon carcinomas displayed a significant increase in bamacan/SMC3 steady state mRNA levels (Fig. 4C). In the colon carcinomas, bamacan mRNA levels were 3.7 Ϯ 0.4 (mean Ϯ S.E.) higher than matched control tissues (p Ͻ 0.0002 by paired Student's t test) (Fig. 4D).
Bamacan Is Overexpressed in Intestinal Tumors of APCdeficient Min/ϩ Mice-There is convincing evidence that most human colon carcinomas exhibit loss of heterozygosity at the Apc locus coding for the APC tumor suppressor gene (16). In mice, a non-sense mutation (Min allele) of the Apc gene causes the development of multiple intestinal neoplasia (Min/ϩ) (17). As in humans, the adenomas in Min/ϩ mice show loss of the wild type Apc allele. Therefore, we sought to establish whether bamacan is also overexpressed in adenomas from Min/ϩ mice. Careful examination of five animals of eight months of age revealed that all had advanced intestinal neoplasia covering ϳ60% of the small intestine. In agreement with the data presented above, there was a ϳ4-fold elevation of bamacan/SMC3 expression in the tumors (Fig. 4, E and F). This elevation was specific inasmuch as no difference in bamacan/SMC3 mRNA levels was detected in the proximal or distal colon, or in the testis, when compared with the levels expressed in the same tissues of sex-and age-matched Apc ϩ/ϩ C57BL/6J mice. The increased expression of bamacan mRNA in the intestinal tumors was confirmed by Western immunoblotting, which showed a ϳ4-fold increase of bamacan protein in the neoplastic tissues (Fig. 4G). These findings support the concept that an APC-related pathway may mediate bamacan up-regulation in colon tumors. DISCUSSION The results of this study strengthen the notion that bamacan/SMC3 may play an important role in tumorigenesis (6). The degree of gene activation that leads to cell transformation in normal mouse cells is comparable with that observed in a series of colon carcinomas cell lines and in the majority of human colon carcinoma specimens. The average 4-fold increase supports the idea that bamacan/SMC3 biosynthesis is tightly regulated in normal cells and that an imbalance in the amounts of this protein can directly affect the transformation program.
Several functional roles have been assigned to eukaryotic bamacan/SMC3 for which it is possible to envision mechanisms linking its overexpression to cell growth disregulation. The involvement of this protein in chromatid cohesion during metaphase points to a first mechanism (18). The protein is part of FIG. 2. Overexpression of bamacan/ SMC3 causes transformation of Balb-c 3T3 fibroblasts. A, Northern blotting of total RNA extracted from clone 6 (a G418-resistant but lacking bamacan/ SMC3 expression) and two overexpressing clones. For loading comparison, the ethidium bromide-stained ribosomal RNA (rRNA) is shown in the lower panel. B, growth in soft agar as determined after 21 days of growth. C, growth curve of three individual clones as indicated. D, focus assay of clones as in A. The wild type was identical to clone 6 (not shown). E, morphological changes and lack of contact inhibition in cells overexpressing bamacan/ SMC3. F, Western immunoblotting of total cell extracts (ϳ20 g of protein each) of clone 6 (lane 1) and clone 9 (lane 2) using a rabbit antibody raised against Pro 1137 -Ser 1157 region of murine bamacan. the cohesin multimeric protein complex that prevents the premature separation of sister chromatids (12). This process occurs in an orderly fashion during anaphase under the control of the cell cycle machinery and, if improperly executed, could result in the segregation of both sisters to the same cell thereby generating an aneuploid karyotype (19). Bamacan/SMC3 is also a necessary component, together with SMC1, of a multimeric complex named RC1 that has DNA recombination/renaturation, DNA ligase, and DNA polymerase activities (13). The enzymes act in concert to repair a gapped or deleted DNA. These different functions of bamacan/SMC3 are mediated by its ability to bind palindromic DNA sequences through its C terminus and by allowing the formation of protein-DNA structures that are accessible to the action of DNA-modifying enzymes (20,21).
The functional significance of proteoglycan core protein in the nucleus and at the same time extracellularly eludes a simple interpretation. Proteoglycans can affect cell growth and differentiation by acting through different signaling pathways (22). Increased levels of proteoglycans with altered composition have been reported in human tumors, primarily those of epithelial origin, such as breast, colon, and lung. Bamacan proteoglycan has been shown to be a normal constituent of the basement membranes in several tissues (3) and is, thus, plausible that its secretion is a physiological event. Whether abnormal accumulation of extracellular bamacan occurs during tumor formation and the exact extracellular function of this protein needs to be established in future studies.
In conclusion, our results support the concept that overexpression of bamacan/SMC3 alone is sufficient to initiate cell transformation in normal fibroblasts. Thus, abnormal expression of bamacan/SMC3 gene may disrupt the formation of the multimeric protein complexes within the nucleus which could directly contribute to the genesis of a transformed phenotype.