Proc. Natl. Acad. Sci. USA Vol. 90, pp. 679-682, January 1993 Medical Sciences Pleiotrophin transforms NIH 3T3 cells and induces tumors in nude mice (protooncogene/growth factor/transformation) ANIL K. CHAUHAN*, YUE-SHENG LI*, AND THOMAS F. DEUEL*tt Departments of *Medicine and tBiochemistry and Molecular Biophysics, Jewish Hospital at Washington University Medical Center, 216 South Kingshighway, St. Louis, MO 63110 Communicated by William H. Daughaday, October 13, 1992

ABSTRACT The pleiotrophin (TN) gene (Ptn) encodes an virus 40 (SV40) early promoter (12) transforms NIH 3T3 18-kDa protein that is highly conserved among mammalian cells, as determined by four independent criteria. The results species and that functions as a weak mitogen and promotes establish that PTN is another member ofa family ofcytokines neurite-outgrowth activity in vitro. To further investigate the with oncogenic potential (13, 14) and indicate that PTN has role PTN plays in regulating cell growth, we overexpressed the the ability to profoundly influence cell growth. bovine PTN cDNA and now show that PTN phenotypically transforms NIH 3T3 cells, as evidenced by increased cell number at confluence, focus formation, anchorage-indepen- MATERIALS AND METHODS dent growth, and tumor formation in the nude mouse. The Plasmids. The bovine PIN cDNA was cloned in the results demonstrate that the Ptn gene has the potential to eukaryotic expression vector pAGE103 (15). A 710-base-pair regulate NIH 3T3 cell growth and suggest that PTN may (bp) fiagment containing 77 bp of the 5'-untranslated region, influence abnormal cell growth in vivo. the 504-bp coding sequence, along with 129 bp of the 3'- untranslated region was cloned into the Kpn I site down- Pleiotrophin (PTN) is an 18-kDa heparin-binding protein that stream from the SV40 early promoter (SV40 ori); the resul- was purified as a weak mitogen from bovine uterus (1) and as tant plasmid was designated pPTN-1 (Fig. 1A). The vector a neurite outgrowth-promoting activity from neonatal rat consists ofbasic elements, the origin ofreplication, the SV40 brain (2, 3). We have cloned the human, bovine, and rat early promoter, and a polyadenylylation splicing signal. In cDNAs encoding PTN (3). The predicted gene products are addition, pAGE103 has multiple cloning sites and the neo- extraordinarily conserved and share 55% identity in amino mycin-resistance gene driven by the thymidine kinase pro- acid sequence with the predicted sequence of a retinoic moter. A second plasmid designated PBSis-1 was con- acid-induced differentiation factor (4, 5) and 49o identity structed by amplifying an =900-bp fragment consisting of with the chicken retinoic acid-induced heparin-binding pro- v-cis coding sequence with 48 nucleotides 5' to the codon tein recently purified from chicken embryo (6). Transcripts of initiating the N terminus and 120 nucleotides 3' to the codon the Ptn gene are expressed in a restricted pattern and are of the C terminus. This amplified fragment was cloned into highly regulated during mouse development (ref. 3; H.-J. Yeh the multiple cloning sites of plasmid pAGE103; this plasmid and T.F.D., unpublished work). The different activities of was used as a positive control for focus-formation assays. PTN, the striking conservation of amino acid sequence Isolation of RNA and RNA Blots. Total cellular RNA was among mammalian species, and the developmental regulation isolated by the guanidium thiocyanate method (16). Fifteen of the Ptn gene suggest that this gene may have important micrograms oftotal RNA was subjected to electrophoresis in roles in normal growth and development. a 1% denaturing agarose gel containing 6% (wt/vol) formal- Although PTN originally was purified on the basis of its dehyde and 40 mM 4-morpholinepropanesulphonic acid mitogenic activity (1), controversy has arisen concerning the (Mops), pH 7.0. RNA was blotted onto a nylon membrane growth-promoting activity of PTN. PTN purified in other (Micron Separations, Westboro, MA) and probed with a laboratories was devoid of mitogenic activity when using a 32P-labeled cDNA fragment encoding the entire Ptn gene number of different assays with several different target cell product. The quantity of RNA was measured by optical lines (7-9). Recently, however, acidic conditions and storage density at 260 nm, whereas the quality of the RNA was of PTN were reported (10) to result in loss of mitogenic checked by ethidium bromide staining before transfer. activity, suggesting a basis for the loss of mitogenic activity Prehybridization and hybridization were done under strin- previously seen during purification (1, 3). Furthermore, PTN gent conditions at 420C [50% (vol/vol) formamide/5x stan- was shown to be mitogenic when tested with bovine brain dard saline/citrate (SSC)/Sx Denhardt's solution/0.1% capillary endothelial cells and angiogenic in the rabbit cor- SDS/yeast tRNA at 0.1 mg/ml/50 mM sodium phosphate, pH neal-pocket assay (10). PTN is also expressed in high levels 7.0]. For hybridization, labeled cDNA probe at 1 x 106 in melanomas that are highly vascularized and supports the cpm/ml was added. After hybridization, the filter was growth of SW13 cells in soft agar (11), suggesting that PTN washed twice in 0.2x SSC/0.1% SDS at 420C, followed by has significant growth-promoting activity in different sys- incubation in the same solution for 1 hr at 650C. tems. Cells and Transfections. Mouse NIH 3T3 fibroblasts were The present experiments were designed to further charac- maintained in Dulbecco's modified Eagle's medium terize the growth-promoting potential of PTN and to suggest (DMEM)/10% fetal bovine serum. Plasmids pAGE103, mechanisms underlying the potential ofPTN to influence cell pPTN-1, and PBSis-1 were transfected into NIH 3T3 cells by growth. In this communication, we show that overexpression the calcium phosphate method (17). Transfectants were ei- of the bovine PTN cDNA (3) under regulation of the simian ther selected with G418 at an active strength of400 pg/ml 24 hr later or fed with normal growth medium. The medium was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: PIN, pleiotrophin; SV40, simian virus 40. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

679 Downloaded by guest on October 2, 2021 680 Medical Sciences: Chauhan et al. Proc. Natl. Acad. Sci. USA 90 (1993)

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FIG. 1. Expression of PTN. (A) Schematic diagram of plasmid pPTN-1. A 710-bp cDNA fragment was cloned in a Kpn I site in plasmid pAGE103 downstream from the SV40 early promoter. tk, Thymidine kinase; ori, origin of replication; Km, kanamycin (Sigma). (B) RNA blot of total RNA prepared from various cells and probed with full-length cDNA encoding the PTN gene product. Lanes: 1, NIH 3T3 cells; 2, pAGE103-transfected cells; 3, pPTN-1-transfected cells; 4, pNM-1 cells; these were derived as a line from the tumors obtained in nude mice after injection of pPTN-1-transfected cells. changed every third day until colonies appeared. The colo- RESULTS nies from pPTN-1-transfected cells were pooled, and the as PTN-1. To seek a role ofthe Ptn gene in growth regulation, NIH 3T3 derived cell line was designated cells were transfected with the construct pPTN-1 cloned in Focus Formation, Soft Agar Assay, and Tumor Formation in pAGE103 (Fig. 1A) driven by the SV40 early promoter. Nude Mice. To assay focus formation, cells were transfected Positive transfectants were selected with G418. RNA blot with various concentrations of DNA in parallel plates, as analysis with the bovine PTN cDNA insert (3) as a probe was described above. The media were changed every third day. used to establish that the transfected cells (PTN-1 cells, see For evaluating foci per gg ofDNA, a parallel set ofplates was Materials andMethods) overexpressed the PTN mRNA (Fig. transfected; one set was selected for neomycin resistance to 1B). In PTN-1 cells the anticipated 1.6-kilobase (kb) PTN calculate transfection efficiency, and the second set was mRNA (Fig. 1B, lane 3) was readily detected. Under identical selected to determine the number of foci to measure trans- conditions, PTN transcripts were not detected in parent NIH forming efficiency. After 2-3 weeks, cultures were stained 3T3 cells (Fig. 1B, lane 1) or in control vector pAGE103- with crystal violet and photographed; the colonies were then transfected cells (Fig. 1B, lane 2). A second cell line, desig- counted. In addition, pBSis-1 (containing the v-sis coding nated as PNM-1, was also used in these experiments. PNM-1 region, see above) was also transfected. Identical assays was derived from tumors in nude mice at sites injected with were done by using both constructs to compare the trans- 2 x 106 PTN-1 cells (see Material and Methods). The Ptn formation efficiency of both of these genes. gene transcripts also were seen in the tumors derived from For soft agar assays, 105 of the pooled pPTN-transfected PNM-1 cells, establishing that Ptn transcripts continue to be cells were suspended in 2 ml of 0.35% (wt/vol) agar contain- expressed at significant levels in these tumors. The cells ing DMEM/20% fetal bovine serum and overlaid onto a 0.5% derived from tumors were resistant to G418, confirming the (wt/vol) agar solution in 35-mm plates. Colonies appeared stable integration of plasmid pPTN-1. microscopically after 10 days and became visible to the naked Growth Properties of PTN-1 Cells. The growth rate of eye after 21 days of incubation. PTN-1 cells was estimated from cultures plated at a density Tumor formation in 6-week-old athymic nude mice (strain of 1 x 105 cells per 35-mm dish. PTN-1 cells increased in CDI Nu/Nu; Charles River Breeding Laboratories) was number nearly 1.5-fold relative to pAGE103-containing cells tested by injecting s.c. 2 x 106 cells suspended in 200 ,ul of after 24 hr and >2-fold after 48 hr. The PTN-1 cells were sterile phosphate-buffered saline at multiple sites. To estab- clustered, and cell numbers became difficult to quantitate 3 lish cell lines derived from these tumors, small pieces of days after seeding. At confluence (4 days), PTN-1 cells had tumors from the center of the tumor mass were aseptically grown to a density estimated at 2-fold higher than in control removed, chopped into fine pieces, and extracted twice for 30 cultures, and the cells had developed a highly refractile, min each with 0.25% trypsin/DNase I at 100 units/ml/ spindled-shaped appearance. When these cells were main- hyaluronidase at 100 units/ml mixture. The cell suspension tained for an additional 12-14 days, foci were readily ob- was centrifuged at 1000 x g, washed two times with Dul- served (Fig 2 B and D), whereas foci were not found in becco's modified Eagle's (GIBCO)/F-12 medium without cultures of control NIH 3T3 cells and of pAGE103 (vector serum, resuspended in Dulbecco's modified Eagle's/F-12 control)-transfected cells (Fig. 2 A and C). In a second set of medium/l0o fetal calf serum, and allowed to grow under 5% experiments, an average of 435 G418-resistant colonies were CO2 at 370C. The cell line derived from these tumors was observed per pmol of DNA transfected, 20% of which designated as PNM-1. resulted in focus formation at 3 weeks. In v-sis-transfected Downloaded by guest on October 2, 2021 Medical Sciences: Chauhan et al. Proc. Natl. Acad. Sci. USA 90 (1993) 681 resistant cells and clonally selected cells from soft agar independently were injected (2 x 106 cells) s.c. into nude mice. Tumors were observed 21 days after injection when cells from pooled G418-resistant colonies were used and 13-14 days after injection of soft agar cloned pPTN-1- containing cells (Fig. 3D). Each of three sites injected in each of 10 nude mice developed tumors; tumors were not found in nude mice injected with NIH 3T3 cells or with pAGE103- containing cells at 21 days. The tumors metastasized exten- sively into distant tissues, including liver, brain, and lung (data not shown). When sections oftumors were analyzed by in situ hybridization with a 35S-labeled RNA probe from the bovine PTN cDNA, a hybridization signal was observed in histologically identifiable tumor cells; PTN transcripts were seen with the antisense probe but were not seen with the VI~~~~~~~~~~~~I sense probe (H.-J. Yeh and T.F.D., unpublished work). As noted above, a cell line (PNM-1) derived from these tumors FIG. 2. Morphology of ppTN-l-transfected cells and focus for- expressed high levels of the PTN mRNA by RNA analysis mation in PTN cDNA-transfected NIH 3T3 cells. (A) NIH 3T3 cells (Fig. 1). These results confirm that the tumor cells continue transfected with eukaryotic expression vector pAGE1i3. (X5.) (B) to overexpress bovine PTN transcripts. Cells transfected with plasmid pPTN-l (see Fig. 1). (x5.) (C) Focus To confirm further the oncogenic potential of the Ptn gene, formation in the NIH 3T3 cells seen 12-14 days after transfection with plasmid pAGE103. (D) Focus formation in the NIH 3T3 cells normal rat kidney (NRK) cells were transfected with the seen 12-14 days after transfection with plasmid pPTN-1. original bovine PTN cDNA in the mammalian expression vector pMAMneo-blue (Clontech). Phenotypic transforma- cells, 75% of the G418-selected colonies formed foci, sug- tion was readily observed in NRK cells transfected with the gesting that Ptn is nearly 27% as efficient as v-sis as a PTN cDNA but was not seen in cells transfected with vector transforming gene. alone (data not shown). Colony formation in soft agar was used to investigate anchorage-independent growth. Visible colonies were DISCUSSION readily detected in PTN-1 cell cultures within 10-12 days (for details, see Fig. 3B); the colonies were prominent at 3 weeks. Overexpression of the bovine PTN cDNA in NIH 3T3 cells At 3 weeks, >95% of the pPTN-1-containing cells plated in results in the transformed phenotype, as judged by four soft agar formed colonies with a minimum of 10 cells in each independent criteria, including increased cell numbers at colony, and, nearly 75% ofthe colonies formed contained 100 confluence, focus formation, anchorage-independent or more cells per colony. Colonies were not seen in cultures growth, and tumor formation in nude mice. The results with untransfected NIH 3T3 cells, although 1-5% of the establish that overexpression of the Ptn gene profoundly pAGE103-containing cells formed microscopic colonies in influences cell growth and is transforming by standard cri- repeated experiments (Fig. 3A). The colonies ofPtn-express- teria. The transforming efficiency of the Ptn gene in these ing cells in soft agar were consistently unusual in appearance; experiments is =27% of the transforming efficiency of the there was external budding from the parent colonies and also potent v-sis gene (18); the transforming efficiency of Ptn is budding within the larger masses of cells (Fig. 3C). Remark- slightly higher than that of the pro-epidermal growth factor ably, budding also was seen in the tumors that formed when gene (19). Tumors arising at the injection sites ofPTN-1 cells pPTN-1-containing cells were injected s.c. in nude mice (see are highly vascularized, metastasize extensively, and are below). unusual in appearance in that multiple bud-like protuber- Tumor Formation. The NIH 3T3 cells transfected with ances extrude from the tumor surface (Fig. 3). The colonies plasmid pPTN-1 were clonally selected from pooled colonies in soft agar also were unusual in appearance: external bud- of G418-resistant cells (see above) by soft agar cloning and ding was a prominent feature (see Fig. 3). These results expanded (see Materials and Methods). Both pooled G418- suggest that overexpression of Ptn gene promotes a highly unregulated cell growth and that, perhaps, PTN may also serve as an angiogenic factor in tumors. Although the exact mechanism by which PTN transforms NIH 3T3 cells is unclear, the unusual appearance of PTN-1 cell tumors in the nude mouse suggests that overexpression of the Ptn gene may cause a significant cytoskeletal disor- ganization that favors unrestricted growth. As reported (2, 3, 7, 9), in addition to its weak mitogenic activity, PTN also induces neurite outgrowth in vitro, also suggesting that PTN D influences cytoskeletal structure and/or may also function as an adhesion molecule to support both neurite outgrowth and colony growth in soft agar. Recently, PITN was reported (11) to enhance growth of an epithelial cell line (SW-13) in soft agar, which also supports the possibility that PTN promotes tumor growth, in part by interacting with other surface molecules. A parallel in the mechanisms by which PTN and the v-sis gene product transform cells is also suggested by this work. FIG. 3. Soft agar assay and tumor formation in nude mice. (A) the pAGE103-transfected cells in soft agar assay. (x5.) (B) pPTN-1- Thus, identification of v-sis oncogene product as the viral transfected cells. (x5.) (C) Soft agar colony. The growth of cells homologue of the B-chain of platelet-derived growth factor within the colonies and budding cell is peculiar. (x20.) (D) Tumors suggested that when growth factor genes are overexpressed, in nude mice. transformation may occur by an autocrine mechanism (for Downloaded by guest on October 2, 2021 682 Medical Sciences: Chauhan et al. Proc. Natl. Acad. Sci. USA 90 (1993) review, see refs. 20 and 21). Targeting ofgrowth factors to the 5. Tomomura, M., Kadomatsu, K., Matsubara, S. & Muramatsu, secretory pathways also appears to be requisite for transfor- T. (1990) J. Biol. Chem. 265, 10765-10770. mation by growth factor genes (22). For PTN, sequences 6. Vrios, P., Duprez, D., Caer, J. L., Courtois, Y., Vigny, M. & encoding the signal peptide were included in the cDNA used Laurent, M. (1991) Biochem. Biophys. Res. Commun. 175, to transform NIH 3T3 cells, and thus PTN enters the secre- 617-624. tory- pathways in the transformed cells in these experiments. 7. Kuo, M.-D., Oda, Y., Huang, J. S. & Huang, S. S. (1990) J. PTN also contains a very basic, lysine-rich C terminus, Biol. Chem. 265, 18749-18752. similar in amino acid sequence to the sequences at the C 8. Bohlen, P., Muller, T., Gautschi-Sova, P., Albrecht, U., Ra- sool, C. G., Decker, M., Seddon, A., Fafeur, V., Kovesdi, I. terminus of the platelet-derived growth factor B chain, the & Kretschmer, P. (1991) Growth Factors 4, 97-107. platelet-derived growth factor A chain, and vascular endo- 9. Hampton, B. S., Marshak, D. R. & Burgess, W. H. (1992)Mol. thelial growth factor/vascular permeability factor, in which Cell. Biol. 3, 85-93. basic C-terminal domains may function as a retention se- 10. Courty, J., Dauchel, M. C., Caruelle, D., Perderiset, M. & quence for the endoplasmic reticulum (23-25). Thus, PTN Barritault, D. (1991) Biochem. Biophys. Res. Commun. 180, could interact with a receptor during the processing of both 145-151. PTN and its receptor, as appears to be the case with v-sis (20, 11. Wellstein, A., Fang, W., Khatri, A., Lu, Y., Swain, S. S., 21, 26), and this interaction could be responsible for or Dickson, R. B., Sasse, J., Riegel, A. T. & Lipman, M. E. contribute to the transforming potential of PTN. (1992) J. Biol. Chem. 267, 2582-2587. The potential of PTN to induce transformation indicates 12. Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289. that PITN has potent growth-regulatory properties, as origi- 13. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., nally suggested (1, 3). The developmental regulation of the Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J. S. & Deuel, T. F. (1983) Nature (London) 304, Ptn gene, the remarkable conservation of predicted PTN 35-39. amino acid sequences among the human, bovine, and rat 14. Deuel, T. F., Huang, J. S., Huang, S. S., Stroobant, P. & species (3), and the -50% identity of predicted amino acid Waterfield, M. D. (1983) Science 221, 1348-1350. sequences with those of the developmentally regulated reti- 15. Mizukami, T. & Itoh, S. (1987) J. Biochem. (Tokyo) 101, noic acid-induced differentiation factor MK- and retinoic 1307-1310. acid-induced heparin RIHB-encoding genes (5, 6) suggest 16. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, that the Ptn gene family may be important in development as 156-159. well. Thus, we conclude that PTN has the potential to play 17. Chen, C. & Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752. a key role in both normal and abnormal proliferative states in 18. Beckmann, M. P., Betsholtz, C., Heldin, C.-H., Westermark, vivo. B., Di Marco, E., Di Fiore, P. P., Robbins, K. C. & Aaronson, S. A. (1988) Science 241, 1346-1349. The overall contributions of the first two authors were equal, and 19. Heidaran, M. A., Fleming, T. P., Bottaro, D. P., Bell, G. I., their names are therefore listed alphabetically. We thank Drs. De Fiore, P. P. & Aaronson, S. A. 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