INTRODUCTION

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Oncogenic conversion of a normal cell into a tumor cell requires multiple genetic alterations (12). Of particular interest is the fact that mutations in both ras oncogenes (3) and the p53 tumor suppressor gene cooperate in transformation of mammalian cells (11). Mutations in both ras and the p53 gene were also found at high frequencies in a variety of human cancers, including those of the colon, lung, and pancreas (2, 18). It has been proposed that both p53 and Ras function, whether directly or through other signaling molecules, to control expression of genes that are important for cell growth and differentiation (13, 17, 37). To this end, several ras target genes (10) and p53 target genes, including those encoding p21/CIP1/WAF1, an inhibitor of G₁ cyclin-dependent kinase (9); Mdm-2, a negative regulator of p53 (1); GADD45, a protein involved in DNA repair (36); and Bax, which promotes apoptosis (28), have been identified. Most of these genes, except p21/CIP1/WAF1, which was cloned by subtractive hybridization, were identified by the candidate gene hypothesis. Recently, more p53 target genes have been isolated by the differential display technique, including those coding for cyclin G (31); MAP4, a microtubule-associated protein negatively regulated by p53 (29); and PAG608, a novel nuclear zinc finger protein whose overexpression promotes apoptosis (14). Functional characterizations of these genes have shed light on the role of p53 in cell cycle control and apoptosis. However, genes that mediate tumor suppression activity by p53 remain elusive.

The fact that neither the inactivation of p53 nor the activation of Ras alone is able to transform primary mammalian cells (34), whereas both mutations together can do so, suggests that genes regulated by p53 and Ras cooperate in upsetting normal cell growth control cells (11). Using differential display (22), we set out to identify genes whose expression is altered by both mutant ras and p53 by comparing the mRNA expression profiles of normal rat embryo fibroblasts (REFs) and their derivatives transformed by either a constitutively inactivated or a temperature-sensitive mutant p53 in cooperation with the activated H-ras oncogene (11, 27). In this report we describe the identification and give a functional characterization of rCop-1, a gene whose expression is abolished by cell transformation. By sequence homology, rCop-1 was found to belong to an emerging cysteine-rich growth regulator family called CCN (which stands for connective-tissue growth factor [CTGF], CEF10/Cyr61, and Nov) (4). Here we show that rCop-1 may represent a novel class of CCN family proteins based on its unique cell cycle expression pattern, its lack of the C-terminal (CT) domain conserved in all CCN proteins, its loss of expression in all transformed cells analyzed, and its ability to confer cytotoxicity to the transformed cells.

	MATERIALS AND METHODS

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Cell culture. All mouse cells and REFs and their derivatives, Rat1, Rat1(ras), T101-4, A1-5, and A1-5/F1, were routinely grown in Dulbecco's modified Eagle medium (Life Technologies, Inc., Grand Island, N.Y.) with 10% fetal bovine serum (HyClone, Logan, Utah) and 1% penicillin-streptomycin (Life Technologies, Inc.) at 37°C with 10% CO₂. CRIP and 2 retroviral packaging cells were maintained in the same condition as described above except 10% bovine calf serum (HyClone) was used instead of 10% fetal bovine serum.

RNA isolation, differential display, and Northern blot analysis. For differential display analysis, REFs (passages 4 to 6) and their transformed derivatives T101-4 and A1-5 were cultured in parallel under identical conditions and grown to 70% confluence before their RNAs were isolated. RNA isolation, differential display, and Northern blot analysis were carried out essentially as previously described (23). Total RNA isolated from the cells was treated with DNase I by using the MessageClean kit (GenHunter, Nashville, Tenn.) before being used for differential display. Differential display was performed by using the RNAmap kit (GenHunter).

Construction and screening of cDNA library. Total RNAs were isolated from REFs as previously described (22) and then further purified by poly(A) selection by using the polyATract mRNA isolation system (Promega, Madison, Wis.). The lambda ZAP II Vector/Gigapack cloning kit (Vector Laboratories, Inc., Burlingame, Calif.) was used to construct the cDNA library following the instructions provided by the manufacturer. A total of 500,000 plaques were screened for full-length rCop-1 cDNA with -³²P-labeled cDNA probe from differential display. Positive plaques were excised as phagemids and sequenced by the molecular biology core facility of the Dana-Farber Cancer Institute.

Construction of recombinant plasmids for rCop-1 expression. A 790-bp BamHI restriction fragment and a 775-bp BspHI-BamH restriction fragment containing the entire coding region of rCop-1 were generated by PCR using the cloned rCop-1 cDNA as a template. Two targeting constructs, a 775-bp BspHI-BamHI restriction fragment and a 790-bp BamHI restriction fragment, both containing the full coding region, were subsequently inserted between the NcoI and BamI sites of retroviral vector pMFG-S and BamI sites of retroviral vector pBabe-Puro and plasmid vector pCMV-Neo/Bam, respectively. All expression constructs were sequenced to ensure the correct coding regions of rCop-1.

Transfection and retroviral infection. Recombinant plasmids pMFG-S-rCop-1 and pBabe-Puro-Cop1 and their vector controls were introduced into the CRIP and 2 viral package cells, respectively, by the standard calcium phosphate precipitation method. Since it does not have any selectable markers, pMFG-S-X was cotransfected with the pCMV-Neo vector at a rate of 10:1 (10 µg of pMFG-S-X: 1 µg of pCMV-Neo) into the packaging cells for the selection of G418 resistance. pBabe-Puro retroviral vector and its derivatives were selected with puromycin upon transfection into the packaging cells to ensure high-titer viral production. Retroviral infection was carried out essentially as described previously (8). Specifically, the target cells for infection were seeded at 1.5 × 10⁴ in each well of the six-well tissue culture dish for 24 h before infection. Cells were infected by incubation for 6 h with the virus-containing medium of the packaging cells in the presence of Polybrene (8 µg/ml). Fresh medium was then added to dilute Polybrene to a concentration of 4 µg/ml. After an overnight incubation, infected cells were washed twice with phosphate-buffered saline (PBS), and fresh medium was added. The phenotypes of the infected cells were either scored after 48 to 72 h of culture or selected with antibiotic resistance for an additional 72 h. Cells resistant to puromycin (2 to 12.5 µg/ml, depending on the target cell lines) were either stained with Giemsa blue or trypsinized for cell count. For checking infection efficiency, virus that encodes a histochemically detectable gene coding for LacZ was used as a control. At 72 h after lacZ virus infection, the cells were fixed in 0.5% glutaraldehyde (Sigma Chemical Co., St. Louis, Mo.) at room temperature for 10 min before being stained with X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) solution (X-Gal [1 mg/ml] in N,N-dimethylformamide-MgCl₂ [1 mM] and 5 mM KFeCN in PBS) for 30 min at 37°C.

Expression, purification of rCop-1 protein, and generation of antibody against rCop-1. A 741-bp BamHI-HindIII fragment, containing the rCop-1 coding region without its N-terminal 23-amino-acid signal peptide, was generated by PCR using cloned rCop-1 cDNA as a template. The primers used were LhisCop1 (5'-GGATCCAGCTGTGCCGGACAC-3') and RhisCop1 (5'-AAGCTTCATTTGCTGAGGATG-3'), respectively. The PCR product was first subcloned into PCR-TRAP vector (GenHunter), and the BamHI-HindIII segment was then excised, purified, and ligated into the corresponding site of the His tag expression vector pQE32 (Qiagen, Chatsworth, Calif.). The recombinant plasmid pQE32-rCop1 was transformed into Escherichia coli TG1 to produce a six-histidine-tagged polypeptide of rCop-1, corresponding to amino acids 24 to 250 upon induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside). The His tag-rCop-1 fusion protein was insoluble when overproduced in the bacteria and thus was purified on a nickel-Sepharose column under denaturing conditions according to the manufacturer's protocol (Qiagen). The purified polypeptide was injected into New Zealand White rabbits to produce anti-rCop-1 polyclonal antibody (HRP Inc., Denver, Pa.).

Immunostaining, immunoblotting, and biotination of cell surface protein. Immunostaining of rCop-1 protein was performed with the Vector ABC kit used according to the manufacturer's recommendation (Vector Laboratories, Inc.). Indirect immunofluorescent staining and biotination of cell surface protein followed by immunoprecipitation (IP)-Western blotting were carried out as previously described, with some modifications (7). For intracellular staining, cells were permeabilized by a 15-min incubation with ice-cold methanol. For surface staining, cells were fixed in 2% paraformaldehyde, which does not permeabilize cells. After rinsing cells with PBS-bovine serum albumin (BSA) buffer, either rabbit anti-rCop-1 or preimmune serum was diluted 1:500 in PBS-BSA buffer containing 5% donkey serum and incubated with the fixed cells for 1 h. After the cells were washed extensively with PBS-BSA, secondary antibody (Cy-3-conjugated donkey anti-rabbit antibody) was added and incubated in the dark for 30 min. Cells were washed extensively, mounted in Vectashield mounting medium, and viewed with a Zeiss Axiophot microscope and a Photometrics CE200A charge-coupled device camera and IP Lab spectrum software. For cell surface staining, cells were fixed for immunoblotting, scraped off the tissue culture plates in extraction buffer (1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 0.1% mercaptoethanol in PBS), and lysed by sonication. Protein concentration was determined with a protein assay kit (Bio-Rad Laboratories, Hercules, Calif.). Aliquots of 150 µg of each protein extract or 150 µl of conditioned medium after concentration were separated by electrophoresis on a sodium dodecyl sulfate-12% polyacrylamide gel (National Diagnostics, Atlanta, Ga.) and blotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.). For Western blot analysis, a 1:2,000 dilution of both rCop-1-specific antibody and the horseradish peroxidase (HRP)-coupled secondary antibody was used. Reactive proteins were visualized with an ECL kit (Amersham, Little Chalfont, Buckinghamshire, England) used according to the manufacturer's protocol.

Fluorescence-activated cell sorting (FACS) analysis. Cells alone or infected with either pBabe-rCop-1 or pBabe vector control retroviruses were selected with puromycin (10 µg/ml) 24 h after infection. Puromycin-resistant cells were washed with PBS and trypsinized after 24 and 48 h of drug selection. After being washed again with cold PBS, the cells were fixed with 67% ethanol at 4°C for 2 h. Cell nuclei were stained with propidium iodide (Sigma Chemical Co.). Histograms of cellular DNA content were measured by quantitative flow cytometry using a FACSCalibur workstation (Becton Dickinson, Mountain View, Calif.).

Tumorigenicity analysis. A 790-bp BamHI restriction fragment containing the entire coding region or rCop-1 cDNA was inserted into the BamI site of the pCMV-Neo/Bam plasmid expression vector. The recombinant plasmid and the vector control were then introduced into the transformed cells by cotransfection with a hygromycin-resistant plasmid, pSV-Hygro. Hygromycin-resistant colonies were clonally purified and expanded, and 5 × 10⁴ cells were injected into athymic NIH nu/nu mice (Taconic, Germantown, Pa.) for tumorigenicity analysis.

	RESULTS

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Loss of rCop-1 expression in cell transformation by mutant p53 and activated H-ras. To identify the molecular alterations during cell transformation, we utilized the differential display method (22, 23) to compare the mRNA expression profiles of normal REFs and their derivatives, A1-5 and T101-4, that were transformed by the cooperation of a dominant negative mutant p53 tumor suppressor gene and an activated H-ras oncogene (27). A1-5 contains a temperature-sensitive mutant, p53(V135), which allows conditional manipulation of p53 function, whereas T101-4 contains a constitutive mutant, p53, with an in-frame decameric insertion between codons 215 and 216 (27). In a four-way comparison of normal REF, T101-4, and A1-5 at nonpermissive (37°C or above) and permissive (32.5°C) temperatures, using 120 combinations of primers which have about 80% coverage of expressed mRNAs (25), 3 genes whose expressions became lost and 12 genes whose expressions were dramatically induced by transformation were identified (24; unpublished results). A cDNA species designated rCop-1 was identified as one of the three genes whose expression became lost after cell transformation (Fig. 1). rCop-1 expression was not restored in the A1-5 cell line at the permissive temperature at which p53 regains its functional conformation (Fig. 1).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Identification of loss of rCop-1 expression following cell transformation. (A) Total RNAs from REFs of passage six (lane 1) and their derivatives transformed by mutant H-ras with either a constitutively inactivated p53 (T101 cells) (lane 2) or a temperature-sensitive mutant, p53 (A1-5 cells), at nonpermissive (lane 3) and permissive (lane 4) temperatures were compared by differential display as described in Materials and Methods. (B) The reamplified rCop-1 probe from the differential display was cloned and used to confirm its differential expression by Northern blot analysis. The lower panel shows the same blot probed with 36B4 as a loading control. Lanes are as described for panel A.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   cDNA sequence of rCop-1. The cloned 418-bp rCop-1 cDNA probe from differential display was used to screen a cDNA library of REFs. The positive clones were sequenced. The longest rCop-1 cDNA (1.7 kb) encodes a predicted polypeptide of 250 amino acids, with a hydrophobic N-terminal signal peptide sequence (the predicted cleavage site is marked by the arrowhead). The 418-bp rCop-1 cDNA probe isolated by differential display is located at the 3' end of the full-length cDNA sequence as predicted, and the primers used for differential display are underlined, with mismatches between the mRNA sequence and the arbitrary primer marked by dots.

rCop-1 is homologous to members of the CCN family of growth regulators. Homology search by computer revealed that rCop-1 is homologous to members of a family of cysteine-rich growth factors called CCN (4). The overall homology is about 40%, while the identity in regions such as the insulin-like growth factor binding domain is as high as 55% (Fig. 3). However, in contrast to all known members of the CCN family of proteins, the predicted rCop-1 protein is shortened by about 100 amino acids, corresponding to the entire CT domain conserved in all other CCN proteins (Fig. 3B and 4). Based on the genomic structure of other members of the CCN family, the cloned rCop-1 cDNA does not contain the exon encoding the CT domain (21). However, without the genomic structure of rCop-1, we could not discern whether the cDNA obtained corresponded to a splicing variant of the gene or whether the rCop-1 gene itself had a loss of exon.

View larger version (55K): [in this window] [in a new window]   FIG. 3.   Homology analysis of rCop-1 and other proteins of the CCN family. (A) The amino acid sequence alignment was prepared by using the Pretty Plot program from Genetics Computer Group sequence analysis software. hctgf and mcyr61 stand for human CTGF and mouse CYR61, respectively. (B) Schematic representation of conserved modular domains of CCN family proteins (4) and rCop-1. Note that rCop-1 does not contain the CT domain conserved in all other CCN proteins. Abbreviations: IGF-BP, IGF binding module; VWC, Von Willebrand factor type C repeat; T, thrombospondin type 1 repeat; and CT, CT module.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Expression of rCop-1 mRNA during G0-to-S transit. (A) The normal BALB/c 3T3 mouse fibroblast cell line A31 was synchronized by serum starvation. Cellular DNA synthesis was measured by [3H]thymidine incorporation following restimulation with serum (26). Northern blot analysis was used to determine the pattern of rCop-1 expression in comparison with that of cyr61, which is known to be an immediate-early gene. (B) Thymidine kinase (tk) mRNA expression, which is S-phase specific, was analyzed to confirm the synchrony of the cells, while 36B4 was used to assure equal loading of the RNA samples.

The rCop-1 expression pattern is unique in the CCN family of genes after the entry of quiescent cells into the cell cycle. Most of the other CCN family members were shown to be immediate-early genes which are induced when quiescent normal fibroblasts are stimulated by serum growth factors to enter the cell cycle (30, 32, 35). The only previous exception was the Nov proto-oncogene, which was only expressed in quiescent chicken embryo fibroblasts, and serum stimulation caused a down-regulation of Nov message (33). In contrast, rCop-1 was neither expressed in quiescent normal A31 BALB/c 3T3 cells nor immediately induced by serum stimulation (Fig. 4). In fact, the pattern of rCop-1 expression appeared to inversely correlate with that of cyr61, and rCop-1 mRNA only started to peak during S phase when the cyr61 mRNA level had been attenuated. rCop-1 expression was steady in continuously growing A31 cells. This result suggests not only that rCop-1 is structurally distinct but also that its expression may be regulated by a mechanism different from that of cyr61 and other CCN growth regulators.

rCop-1 expression is also lost in MEFs transformed by a variety of means. rCop-1 was isolated by differential display as a gene whose expression became lost after REFs were transformed by mutations in both H-ras and p53. By using a rat cDNA probe, rCop-1 was shown by Northern blot analysis to be expressed in normal BALB/c 3T3 A31 mouse embryo fibroblasts (MEFs) but not in any of their derivatives transformed by a variety of means, including alkylating agents (BPA31 and DA31), Kirsten and simian virus 40 viruses (for KA31 and SVA31 cell lines), and spontaneous mutation (3T12) (reference 26 and Fig. 5). In contrast, cyr61 mRNA did not seem to be differentially expressed between normal and transformed cells (Fig. 5).

View larger version (78K): [in this window] [in a new window]   FIG. 5.   Northern blot analysis of the down-regulation of rCop-1 in murine BALB/c 3T3 cells transformed by a variety of methods. Normal parental A31 cells were compared with chemically transformed (BPA31 and DA31), virally transformed (KA31 and SV31), and spontaneously transformed 3T12 cells. The same blot was probed with the cyr61 cDNA. Ethidium bromide staining of the rRNA is shown as a loading control.

Retroviral gene transfers of rCop-1 expression constructs. Realizing the potential danger of selecting for growth after stable transfection, when working with a gene whose product may inhibit cell transformation we utilized retroviral gene delivery systems to deregulate rCop-1 expression. The pMFG retroviral vector has the advantage of producing helper-free recombinant retrovirus that can infect transformed rodent fibroblasts with 90 to 95% efficiency (8). One does not have to wait for weeks to expand cells from single colonies. Using such a vector, we demonstrated that the infection with rCop-1-expressing retrovirus led to a dramatic decrease in the number of transformed cells within 3 days postinfection (Fig. 6A), in comparison to controls with lacZ retroviral infection and cells without viral infection. Immunohistochemical staining of transformed cells infected with rCop-1-expressing virus showed that most of the dying cells which became rounded and light scattering were positive for rCop-1 protein expression, whereas the surviving, well-attached cells that presumably escaped from viral infection were not stained by rCop-1-specific antibody (Fig. 6A). Furthermore, no rCop-1 mRNA expression was detected on Northern blots of the surviving cells that eventually grew up after the infection with rCop-1 virus.

View larger version (77K): [in this window] [in a new window]   FIG. 6.   Inhibition of transformed cell growth by retrovirally mediated gene transfer of rCop-1 expression. (A) The transformed A1-5 cells were infected with no virus (a), pMFG-lacZ virus (b and c), and pMFG-rCop-1 virus (d and e). The cells were photographed under a microscope 3 days after infection. The efficiency of retroviral infection was determined by X-Gal staining for the expression of -galactosidase in pMFG-lacZ virus-infected cells (c). The surviving cells from rCop-1 viral infection were stained for the expression of rCop-1 by immunohistochemistry using the rCop-1-specific antibody followed by HRP staining (e). Note that the rounded and light-scattering cells stained positive for rCop-1 (black staining). (B) Rat-1 cells and their derivative transformed by oncogenic H-ras were infected with either pBabe-Puro vector or pBabe-Puro-rCop-1 viruses as indicated. The infected cells were selected for puromycin resistance for 72 h. The surviving cells were stained with Giemsa blue and photographed under a microscope. Note that the nontransformed Rat-1 cells infected by both viruses formed a confluent monolayer and stained less strongly than the transformed cells, which grew in multilayers and formed foci.

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Quantification of differential inhibition of transformed cell growth by retrovirally mediated rCop-1 expression. Nontransformed Rat-1 cells and transformed Rat-1(ras), A1-5 and T101-4 cells were infected with either the pBabe-Puro vector control or rCop-1 retroviruses as described in the legend to Fig. 6B. The numbers of puromycin-resistant cells with and without infections were determined from duplicate samples after 72 h of selection with puromycin.

rCop-1 expression in retrovirally infected cells. To substantiate that the differential effect of rCop-1 retroviral infection on the transformed cells is caused by the overexpression of the gene, both the rCop-1 mRNA and protein expression were measured in puromycin-resistant cells following infection and puromycin selection. The result shown in Fig. 8A indicates that rCop-1 expression was detected at both mRNA and protein levels only in the pBabe-rCop-1 retrovirus-infected cells but not in cells infected with vector control virus. Moreover, similar levels of rCop-1 mRNA and protein expression were seen after pBabe-rCop-1 retroviral infection of either Rat-1 cells or their derivative transformed by oncogenic ras, Rat-1(ras). No rCop-1 protein was detected from conditioned medium of Rat-1 cells infected with pBabe-rCop-1 retrovirus (Fig. 8B).

View larger version (44K): [in this window] [in a new window]   FIG. 8.   rCop-1 expression in cells infected with rCop-1 retrovirus. (A) Nontransformed Rat-1 cells (lanes 1 and 2) and oncogenic ras-transformed derivative Rat-1(ras) cells (lanes 3 and 4) were infected with either the pBabe-Puro vector control virus (lanes 1 and 3) or pBabe-rCop-1 virus (lanes 2 and 4) and selected with puromycin. Both Northern blot analysis (upper panel) and Western blot analysis (middle panel) of these cells indicated the expression of rCop-1 after infection with the rCop-1 retrovirus. The lower panel shows the staining of rRNA as a control for equal RNA sample loading. (B) Western blot analysis of rCop-1 expression in conditioned media of cells infected with the recombinant rCop-1 retrovirus. Conditioned media from Rat-1 cells infected with pBabe-Puro vector control (lane 1) or pBabe-rCop-1 viruses (lane 2) and their corresponding cellular protein extracts (lanes 3 and 4) as described in the legend to Fig. 8A were analyzed. Protein extracts (150 µg) or conditioned media (150 µl) after concentration were analyzed for each sample.

rCop-1 overexpression by retroviral infection in transformed cells leads to cell death. To determine if the effect of rCop-1 expression on transformed cells was due to either cell growth inhibition at a particular cell cycle point or killing of the cells, we performed FACS analysis of the DNA content of infected cells after 24 and 48 h of puromycin selection. The result showed that rCop-1 overexpression led to a significant increase in the number of transformed cells with sub-G₁ DNA content, suggesting that the effect of rCop-1 is cell killing rather than cell cycle arrest (Fig. 9). However, nontransformed Rat-1 cells were little affected by the rCop-1 expression, consistent with the findings obtained by cell counts (Fig. 6 and 7).

View larger version (30K): [in this window] [in a new window]   FIG. 9.   FACS analysis of cellular DNA content of cells infected with rCop-1 retrovirus. Nontransformed Rat-1 cells and transformed counterparts Rat-1(ras) or T101-4 cells were infected with either the pBabe control or pBabe-rCop-1 retroviruses as indicated. FACS histogram analysis of puromycin-resistant cells was conducted 48 h following puromycin selection. No attached viable cells were observed after 48 h of puromycin selection in plates which had cells alone without infection with either virus. Note that both transformed cells, but not the nontransformed Rat-1 cells, had a large cell population with a sub-G1 DNA content (<200), indicating DNA fragmentation in these cells.

Subcellular localization of rCop-1 protein expression. Based on the presence of a typical signal peptide sequence at its N terminus, rCop-1 may be a secreted protein, like most of the CCN family of proteins. However, CYR61 was found to be associated mostly with extracellular matrix (ECM) and little was detected in the conditioned medium (38). Using Western blot analysis of subcellular fractions, we showed that rCop-1 protein was associated with the cells overexpressing the rCop-1 gene, but not in their conditioned medium or ECM fractions (Fig. 8; data not shown). To differentiate whether the rCop-1 protein is localized inside the cell or on the cell surface, immunostaining using both cell-permeable (methanol) and nonpermeable (paraformaldehyde) fixation agents was performed. The result indicated that a significant amount of rCop-1 staining was detected on the cell surface, although the majority of overexpressed rCop-1 appeared to be retained in the cytoplasm based on the intensity of the stainings (Fig. 10). It is interesting that although the A1-5 cells overexpressing rCop-1 examined by immunostaining were derived from a clonally purified stable transfectant (Fig. 11), more than half of the cells appeared to have lost their rCop-1 expression during passages in culture, even in the presence of continuous drug selection. Although two hygromycin-resistant colonies overexpressing rCop-1 were obtained and expanded into cell lines, the cell killing effect of rCop-1 overexpression in transformed cells detected by efficient retroviral infection certainly would be hard to measure by stable transfection. This finding seems to be consistent with the rCop-1 gene's being a transformation suppressor, because its loss of expression may afford transformed cells an advantage for survival or growth which is needed to obtain stable transfectants.

View larger version (113K): [in this window] [in a new window]   FIG. 10.   Immunostaining showing unstable rCop-1 expression in A1-5 cells stably transfected with the rCop-1 expression vector and cell surface detection of rCop-1 protein. Exponentially growing clonally purified A1-5/rCop-1 stable transfectants were permeabilized with methanol and stained with either the preimmune serum (B, D, and F) or an equal dilution of the rCop-1 antibody (A, C, and E). The expression of rCop-1 was visualized with either HRP staining using the Vector Stain ABC kit (Vector Laboratories) (A and B) or Cy-3 fluorescently labeled secondary antibody (C and D). rCop-1 antibody produced strong perinuclear staining as well as some vesicular staining in the cytoplasm. Immunofluorescent staining of rCop-1 showing punctate distribution of rCop-1 protein on the cell surface was carried out by fixing cells with 2% paraformaldehyde, which does not permeabilize the cells (E). (F) Phase-contrast view of the same cells. Note: not all cells express rCop-1 (A, E, and F).

View larger version (44K): [in this window] [in a new window]   FIG. 11.   Detection of cell surface rCop-1 and inhibition of tumorigenicity by mixed culture of transformed cells with or without rCop-1 expression. (A) The transformed A1-5 cell line was stably transfected with pCMV-Neo/Bam-rCop-1 and vector control. The expression of rCop-1 mRNA in the parental A1-5 cells (lane 1), vector-transfected cells (lane 2), and rCop-1 expression vector-transfected cells (lane 3) was analyzed by Northern blotting. (B) Cell surface-localized rCop-1 protein was detected by biotination of cell surface proteins followed by IP-Western blot analysis with HRP-streptavidin and affinity-purified rCop-1 antibody. (C) For tumorigenicity analysis, 5 × 104 vector-transfected cells and a clonally purified rCop-1-expressing A1-5 line, which has fewer than 50% of the cells expressing rCop-1 protein (see Fig. 10), were injected subcutaneously into the left and right legs of the athymic mice, respectively. Tumor formation was observed 3 to 4 weeks after the injections. The results shown are representative of three independent experiments.

rCop-1-overexpressing stable transfectants of transformed cells exhibit reduced tumorigenicity. One important question concerning the effect of rCop-1 on transformed cells is whether the protein works in cis or in trans. Since rCop-1 was detected on the cell surface but not in conditioned medium, the protein may function through cell-cell contact. To this end we conducted an in vivo tumorigenicity experiment in which we used the A1-5/rCop-1 transfectant, which had less than 50% of the cells expressing rCop-1 protein, as described above, and compared it with the parental A1-5 cells. A1-5/rCop-1 cells consistently showed markedly reduced tumorigenicity potential in comparison with the parental cells (Fig. 12).

View larger version (62K): [in this window] [in a new window]   FIG. 12.   rCop-1 expression is induced in rodent embryo fibroblasts passaged in culture. Primary REFs and MEFs were prepared from 14.5-day-old Fisher rat embryos and wild-type 129 mouse embryos. The primary embryo fibroblasts were continuously split in a 1/4 ratio (at each passage) when they became confluent. Northern blot analysis of rCop-1 expression was performed with 20 µg of total RNA isolated from REFs (A) and MEFs (B) at different passages as indicated.

rCop-1 is expressed only in the aging normal rodent embryo fibroblasts. To gain more insight into the physiological role of rCop-1, in situ hybridization using whole-mount mouse embryos and Northern blot analysis of major organs of adult rats and mice, including brain, heart, lung, kidney, pancreas, spleen, intestine, stomach, and skeletal muscle, were used to localize the sites of rCop-1 expression. Quite surprisingly, both methods failed to detect any rCop-1 mRNA expression (data not shown). Interestingly, the rCop-1 gene was found to be expressed only when primary REFs and MEFs began to age or became senescent during passage in culture (Fig. 12). This finding originated from a surprising observation that primary REFs and their early passages that we prepared from rat embryos did not express any rCop-1 mRNA and that the REFs used in the initial differential display were from a culture after the tertiary culture.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Here we describe the isolation and characterization of rCop-1, a novel CCN family protein whose expression was completely lost after cell transformation. Functional studies suggest that rCop-1 is a negative regulator for cell transformation based on the following findings. The loss of rCop-1 expression correlates extremely well with cell transformation in culture, since cells transformed by a variety of mechanisms all lost rCop-1 expression. BALB/c A31 and Rat-1 are both immortalized but nontransformed, yet only the former expresses rCop-1. However, both A31 and REFs, the parental cells of Rat-1, lose rCop-1 expression when transformed by a variety of means. This suggests that the loss of rCop-1 expression in cultured rodent fibroblasts may not be necessary for cell immortalization but may be so for cell transformation. Efficient retroviral gene transfer of rCop-1 exhibited a dramatic cytotoxic effect on the transformed cells but had little effect on the nontransformed cells.

The first member of the CCN gene family, CEF10, was identified as a gene induced by the pp60^v-src oncogene (35). Its close relative, cyr61, was cloned independently from normal murine 3T3 cells as an immediate-early gene inducible by serum growth factors (30). Extensive work on CYR61 showed that the protein is associated with the cell surface and the ECM (38). CYR61 functions as a positive cell growth regulator which promotes cell adhesion and potentiates the mitogenic activity of other growth factors such as platelet-derived growth factor and fibroblast growth factor (19). A third member of the CCN family is CTGF, which was isolated as a mitogen and chemotactic agent for fibroblasts from the conditioned medium of cultured human umbilical vein endothelial cells (5). CTGF was shown to be regulated by transforming growth factor  (20). The mouse homolog of the CTGF gene, fisp-12, was also isolated as an immediate-early gene (32). The last known member of the CCN family is the Nov proto-oncogene, which was overexpressed and activated in nephroblastomas induced by myeloblastoma-associated viral infection (16). Myeloblastoma-associated viral infection resulted in fusion of the proviral long terminal repeat region with the N-terminal portion of the nov gene, leading to its overexpression. Furthermore, amino-terminally truncated Nov protein was shown to be sufficient for transforming chicken embryo fibroblasts (17). These studies established the importance of the CNN family in positive cell growth regulation and their involvement in carcinogenesis.

Although rCop-1 exhibits about 40% identity in amino acid sequence to all members of the CCN family of proteins, it does not have the CT domain of about 100 amino acid residues conserved in all other CCN proteins previously identified (Fig. 3 and 4). Although we could not rule out the possibility that the isolated rCop-1 cDNA is a splicing variant which misses the last CT domain-containing exon conserved in all other CCN genes, we deemed this is an unlikely event for the following reasons. Of all seven independent cDNA clones isolated from the REF cDNA library and four clones isolated by 5' rapid amplification of cDNA ends, all clones had the same stop codon for the rCop-1 coding region and the 3' untranslated sequence as shown in Fig. 2. Also, a single message was detected by Northern analysis. Interestingly, several newly isolated heparin-binding growth factors in uterine secreted fluid were found to be truncated forms of CTGF, which essentially are the CT domains of the protein (6). This intriguing finding suggests that the N-terminal two-thirds of CTGF, which is structurally equivalent to rCop-1, is not required for the mitogenic activity or heparin binding but rather may function to modulate the mitogenic activity of the CT domain. This may also explain why the N-terminal truncation of Nov led to cell transformation. Certainly rCop-1 represents a new form of CCN protein, and unlike most of the known CCN proteins it is a negative cell growth regulator with a specificity to the transformed cells. Further structural and biochemical characterizations of rCop-1 may shed light on the biological function of the N-terminal portion of members of the CCN family of proteins.

In addition to its unique structural difference, rCop-1 also differs from the other CCN family members in the pattern of gene expression. While most of the previously identified genes of the CCN family were shown to be immediate-early genes in normal cells, rCop-1 expression peaked at late S phase when the mRNA levels of other members, such as cyr61, began to become extinct. In transformed cells which no longer express rCop-1, cyr61 expression could be still detected, though in most cases it was much reduced. Also, the other CCN gene, CEF10, was induced by oncogenic transformation by v-src. These results suggest that rCop-1 expression is regulated by a mechanism different from that for most CCN family members. The only exception was the Nov proto-oncogene, which was recently identified by differential display as a gene whose expression was repressed by v-Src-mediated cell transformation (33). But unlike rCop-1 or other CCN genes, nov was shown to be expressed only in quiescent cells at G₀. From the pattern of its gene expression, Nov may be a negative cell growth regulator like rCop-1. However, its biological function in suppressing cell transformation remains to be demonstrated.

Since the mechanism of action for most, if not all, CCN proteins remains to be determined and no receptors or interacting proteins have been clearly established, it is difficult to interpret how rCop-1 could be toxic to the transformed cells but not the nontransformed cells. It seems that rCop-1 is not a death gene per se given the fact that tertiary or later passages of REFs and immortalized MEFs which all express the gene are viable. Since the transformed cells may have more genes, other than rCop-1, whose expression has been altered in comparison with their normal counterparts, it is possible that rCop-1 expression becomes incompatible with the reprogrammed gene expression needed for the growth of the transformed cells. rCop-1 did not appear to work as a diffusible secreted factor, but rather may function either within the cells or at the cell surface based on its subcellular localization and the in vivo coculture tumorigenicity experiments. The conditioned medium of rCop-1-overexpressing cells had neither detectable rCop-1 secretion nor any effect on the transformed cells when added in trans (data not shown).

Rather surprisingly, rCop-1 expression was not detected by Northern blot analysis in major tissues of adult rats, including brain, heart, lung, kidney, spleen, muscle, skin, and intestine, nor was its expression detected by in situ hybridization of the whole-mount mouse embryos. Consistent with this is that rCop-1 expression was not detected in primary REFs or MEFs but began to increase dramatically after the third to fourth passages (six to eight doublings) in culture. It is tempting to speculate that rCop-1 represents a type of tumor suppressor gene whose expression is activated only by aging or abnormal growth, which occurs both in vivo and in vitro, such as when primary REFs and MEFs are forced to grow in culture. Nonetheless, the discovery of rCop-1 increases the structural and functional diversity of the CCN family of proteins and further strengthens their important roles in cell growth regulation and tumorigenesis.