Expression of Oncomodulin Does Not Lead to the Transformation Or Immortalization of Mammalian Cells in Vitro

Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro

ANNE-MARIE MES-MASSON*'t, SYLVIE MASSON, DENIS BANVILLE and LORRAINE CHALIFOUR

Biotechnology Research Institute, National Research Council of Canada, 6100 Royahnount Avenue, Montreal, Quebec, Canada H4P 2R2

* Author for correspondence •f Present address: Institut du Cancer de Montreal, 1560 Rue Sherbrooke est, Montreal, Quebec, Canada H2L 4M1

Summary

A recombinant plasmid (pMTONCO) containing able to prolong the life-span of primary mouse the coding sequences for rat oncomodulin under the kidney cells in culture, no equivalent activity was direction of the metallothionein promoter was con- noted when the pMTONCO plasmid was used to structed. pMTONCO was co-transfected with the transfect primary cells. When expressed in Rat-1 pSV2-NEO plasmid into primary mouse kidney cells, oncomodulin did not affect the growth cells or Rat-1 cells using the calcium phosphate properties of these cells, nor did it predispose cells technique and stable transformants were isolated to higher frequencies of oncogenic transformation after selection with G418. Transcription from the to a viral oncogene. We conclude that oncomodulin metallothionein promoter was inducible 'with is neither an immortalizing nor transforming agent heavy metals and produced an oncomodulin- in vitro. specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plas- Key words: oncomodulin, oncodevelopmental protein, mid encoding the polyomavirus T-antigens was transformation.

Introduction is present in tumor tissue of rat hepatomas (MacManus, 1979), rat fibrosarcomas and mouse sarcomas Oncomodulin was initially identified as a novel low (MacManus, 1981a). In addition to its presence in molecular weight calcium-binding protein found in ex- spontaneously occuring rodent tumors, oncomodulin has tracts of rat liver tumors (MacManus, 1979). Direct also been detected in solid tumors following the injection protein sequencing has demonstrated the sequence of the of rodent transformed cell lines into nude mice 108 amino acids that comprise the complete protein (MacManus et al. 1982). The synthesis of oncomodulin (MacManus el al. 1983). Recently, the amino acid has also been observed in the nucleus of virally trans- sequence of rat oncomodulin has been confirmed, with formed normal rat kidney cells in vitro (Durkin et al. the exception of a single amino acid difference, by 1983). The presence of a calcium-binding protein similar sequence analysis of a complete cDNA encoding this to rodent oncomodulin has also been observed in human calcium-binding protein (Gillen et al. 1987). These tumors (MacManus and Whitfield, 1983; MacManus et results, in addition to earlier immunological evidence al. 1984), cell lines derived from human tumors (Pfyffer (MacManus, 198k/), clearly demonstrate that oncomo- et al. 1984), or tumors formed in nude mice following dulin is distinct from previously described calcium- injection of transformed human cell lines (MacManus et binding proteins, but shares sufficient homology to be al. 1982). Recent results indicate that the expression of considered a member of the troponin C superfamily oncomodulin in rat tumor tissue is regulated at the level (MacManus et al. 1983). Oncomodulin shares the of mRNA transcription (Gillen et al. 1987). greatest homology with rat parvalbumin, at both the The role of oncomodulin, as well as its possible nucleic acid and the amino acid level (Gillen et al. 1987; function within the tumor cell, remains unclear. While it MacManus et al. 1983). has been demonstrated that oncomodulin has a calmodu- In rats, oncomodulin is normally expressed extra- lin-like ability to stimulate the hydrolysis of cyclic AMP embryonically in placenta, yolk sac and amnion (Brewer by rat heart phosphodiesterase (MacManus, 19816; and MacManus, 1985). While oncomodulin has never Mutus et al. 1985), and to a much lesser extent rat brain been detected in normal adult rat tissue, interest in this phosphodiesterase (Klee and Heppel, 1984), oncomodu- protein stems mainly from the finding that oncomodulin lin does not generally mimic the activity of calmodulin. Journal of Cell Science 94, 517-525 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 517 Although oncomodulin has been shown to stimulate the morphology and growth patterns of cells in a manner calcium-starved non-neoplastic liver cell DNA synthesis similar to oncogenes. A recombinant clone permitting the (Boynton et al. 1982), the interpretation of this result is expression of rat oncomodulin in mammalian cells was difficult, since oncomodulin is not present in regenerat- constructed and introduced into primary baby mouse ing liver (MacManus and Whitfield, 1983). kidney cells or cells from the established Rat-1 line by Like alpha-fetoprotein (AFP) and carcinoembryonic DNA transfection. Our results indicate that oncomodulin antigen (CEA), oncomodulin appears in both developing does not act as a cellular immortalizing or transforming and neoplastic tissues, and as such is considered an agent in vitro. oncodevelopmental protein (Brewer and MacManus, 1985). While oncodevelopmental proteins may be useful Materials and methods as tumor markers, their role, if any, in the development or progression of carcinogenesis remains unknown. In Recombinant plasmids contrast, oncogenes clearly play a role in neoplasia. At All enzymatic reactions were performed as described by the least two discernable activities have been described for manufacturer. Standard protocols for plasmid DNA purifi- oncogenes in vitro: their ability to immortalize primary cation, agarose gel electrophoresis, DNA fragment purification cells and/or to transform established cell lines in culture and plasmid transfection into bacteria were employed (Maniatis (Rassoulzadegan et al. 1982; Land et al. 1983; Ruley, et al. 1982). 1983). Because oncomodulin appears to be almost ubiqui- Details of the construction of the pMTONCO plasmid are tously present in rodent tumor tissues (MacManus, 1979; described in Fig. 1. The starting plasmid, pPXMT, was MacManus, 1981«; MacManus et al. 1982), and since it originally provided by J. Sambrook (University of Texas Southwestern Medical Center) and was obtained from J. A. has been shown to influence DNA synthesis indirectly Hassell (McMaster University). Apart from plasmid sequences, (Boynton et al. 1982), it was of interest to determine pPXMT contains a 0.6kb (1 kb=103 bases) Kpnl-Bglll frag- whether the expression of oncomodulin could influence ment of the mouse metallothionein I promoter and a

Kpnl

Bam HI Hind I y Bglll

Hind

Hind III Bam HI Kpn I

Hind III

plasmid SV40 VZL oncomodulin • metallothionein polylinker Fig. 1. Schematic representation of the cloning steps involved in the construction of the recombinant plasmid pMTONCO.

518 A.-M. Mes-Masson et al. Bglll (formally a Bgll site) - BamHl fragment (spanning Northern blot analvsis nucleotidcs 5235-2533) of the simian virus (SV40) early region. RNA was extracted from cells as previously described (Chirg- pPXMT was digested with Bglll and Hindlll to remove the win et al. 1979). Since the metallothionein promoter is respon- majority of the SV40 early region, leaving only SV40 sequences sive to heavy metal induction, some cells were maintained for from the ///wdIII-Ba/«HI site (nucleotides 3476-2533), which 2h in metal-supplemented medium (lOmM-zinc chloride, contain, among other sequences, the early SV40 polyadenyl- 1 mM-cadmiun chloride) before RNA extraction. Total RNA ation signal. The digested fragment was ligated to a small (10 fig) was electrophoretically separated in a denaturing BawHI-.W/«dIII polylinker fragment obtained from the plas- agarose/formaldehyde gel, the gel was stained with ethidium mid pGEM-1 (Promega). The resulting recombinant (pPMT) bromide to verify the integrity and concentration of the RNA was purified from Eschericia coli GM150, a dam methylase sample, and the RNA was transferred to nitrocellulose using minus strain. pPMT DNA was cleaved by restriction endonu- standard techniques (Maniatis et al. 1982). Rat oncomodulin clease digestion, and the large Hindlll-Bcll fragment was gel sequences were detected by hybridization to a [32P]CTP- purified. In parallel, a cDNA fragment of rat oncomodulin, labelled RNA probe complementary to the coding sequences of containing a Hindlll linker at nucleotide +10 and a BamHl oncomodulin mRNA sequences. Hybridizations were per- linker past nucleotide +660 (Gillen et al. 1988) was gel purified formed at 60°C in 50% formamide, 50niM-sodium phosphate and ligated to the large Hindlll-Bcll fragment of pPMT. The (pH7.0), 800mM-sodium chloride, 1 mM-EDTA (pH8.5), resulting plasmid, pMTONCO, was partially sequenced to 2.5xDenhardt's solution, 250jugml"1 denatured salmon sperm ensure that no alterations had occurred in sequences surround- DNA and 500 jug ml"1 yeast tRNA. Hybridization mixtures also ing the AUG translation initiation codon of oncomodulin. contained radioactively labelled RNA at 2X106 ctsmin"1 ml"'. Two other recombinant plasmids were also employed in this Excess probe was removed after hybridization by three success- study. pSV2NEO has previously been described (Southern and ive 30-min washes at 60°C in 50mM-sodium chloride, 20 mM- Berg, 1982). The plasmid pSV2NEOSVEBla was a kind gift sodium phosphate (pH7.0), 1 mM-EDTA (pH8.5) and 0.1% from Bernard Massie (Biotechnology Research Institute, (w/v) sodium dodecyl sulfate (SDS). Dried filters were ex- National Research Council of Canada). Briefly, pSV2NEOS- posed, with intensifying screens, at —80°C for 1-5 days with VEBla contains both the large BamHl-Pvul fragments of Kodak XAR-5 film." pSV2NEO and pPSVEl-Bla (Muller et al. 1984), so that the synthesis of neomycin or the synthesis of small, middle and Analvsis of oncomodulin protein large T-antigens of polyomavirus are driven by separate SV40 Newly synthesized proteins were metabolically labelled by early promoter regions. incubating 8xlOs cells/60mm dish in 0.5 ml methionine-free DMEM containing lOO^Ci of [3sS]methionine (800 Ci mmol" ). Immunoprecipitation of labelled oncomodulin pro- Cell culture and transformation tein was performed as previously described (Chalifnur et al. 1986). Affinity-purified goat anti-oncomodulin IgG was kindly All cells were propagated in Dulbecco's modification of Eagle's provided by J.P. MacManus (Division of Biological Sciences, medium (DMEM) supplemented with 10% fetal bovine serum, National Research Council of Canada). Immunoprecipitated gentamicin (50^

Role of oncomodulin in transfonnation 519 B D

— 28S —

18S — I

Fig. 2. Northern blot analysis of RNA from either control or cell lines expressing the rat oncomodulin cDNA. Lane A contains RNA purified from rat hepatomas, and the arrow indicates the position at which endogeneous rat oncomodulin migrates within a 1.4% agarose/formaldehyde gel. Lanes B and C represent RNA isolated from Rat-1 and NEO5 cells, respectively. RNA was isolated from the MTONCO2-7 cell line either before (lane D) or after (lane E) cells were induced with heavy metals. In a similar manner, RNA from the MTONCO2-12 cell line was isolated before (lane F) or after (lane G) heavy metal induction. Note that the oncomodulin cross-hybridizing species in lanes D, E, F and G do not migrate with endogeneous oncomodulin RNA. In addition, the size of the oncomodulin RNA produced by the MTONCO2-7 and MTONCO2-12 cell lines appears to differ by approximately 50-100 nucleotides.

(M and NEO5) cell lines, or from the nine G-418 cell lines • isolated from co-transfection experiments. The latter cell lines were grown in either normal or heavy metal- O supplemented media. Of the original nine cell lines, five O o G-418 cell lines were found to produce detectable levels 1 O •+—' 8 of oncomodulin mRNA, and in each case the synthesis of 111 l~ oncomodulin RNA was inducible by heavy metals (data DC 5 not included). Of these five cell lines, two (MTONCO2- 7 and MTONCO2-12), producing different constitutive levels of oncomodulin mRNA, were chosen for further study. The oncomodulin RNA pattern of expression of -21.5K MTONCO2-7 and MTONCO2-12 is shown in Fig. 2. The presence of oncomodulin mRNA can be seen in each case before heavy metal induction, but the oncomodulin- •••• -14.5K specific mRNA levels increased approximately threefold after induction. The constitutive and induced levels of oncomodulin RNA appear to be higher in the MTONCO2-12 cell line relative to the MTONCO2-7 cell line. The length of the RNA produced in the -6.5 K MTONCO2-7 and MTONCO2-12 cell lines appears to differ by about 50-100 nucleotides. To determine whether the RNA produced in these cell lines could be translated to produce authentic oncomodu- 35 lin protein, [35S]methionine-labelled proteins from con- Fig. 3. Autoradiograph of immunoprecipitated S-labelled trol (Rat-1 and NEO5), MTONCO2-7 and MTONCO2- protein with goat anti-oncomodulin antibody. Control cell lines include the parental (Rat-1) and pSV2NEO-transformed 12 cell lines were immunoprecipitated with anti-oncomo- cell line (NEO5). Although all cell lines appear to contain a dulin antisera. An autoradiograph of the immunoprecipi- 3 14K (K=10 Mr) background protein, only the MTONCO2-7 tated proteins, after separation by SDS-polyacrylamide and MTONCO2-12 cell lines show a specific 12.6K protein gel electrophoresis and fluorography is presented in band that correlates with the expected molecular weight for Fig. 3. Although all cell lines appear to have a 14000A/r authentic rat oncomodulin.

520 A.-M. Mes-Masson et cil. background band that is precipitated by the anti-oncomo- neither the MTONCO2-7 nor MTONCO2-12 cell lines dulin antisera, only the MTONCO2-7 and MTONCO2- displayed significant growth in soft agarose. Finally, 12 cell lines contain a specifically immunoprecipitated while the Rat-ISMLT cell line did not display contact protein of the correct molecular weight for oncomodulin. inhibition and readily formed foci as cells reached conflu- In addition, radioimmuneassays for oncomodulin were ence, the MTONCO2-7 and MTONCO2-12 cell lines performed. While the Rat-1 and NEO5 cell lines con- resembled the parental Rat-1 cell line in their inability to tained undetectable levels of oncomodulin (<25 ng onco- form foci, even in heavy metal-supplemented media. modulin mg"1 total protein) both the MTONCO2-7 and We also wished to address the question of whether the MTONCO2-12 cell lines produced significant amounts expression of oncomodulin, while not by itself a trans- of oncomodulin. In particular, in the absence of heavy forming agent, could predispose a cell to oncogenic metal stimulation of the methallothionein promoter, the transformation. For this purpose the frequency at which MTONCO2-12 cell line produced upwards of 1500 ng 1 different cell lines could be transformed by the plasmid oncomodulin mg" total protein. This level of expression is comparable to previously reported results for the pSV2NEOSVEBla was assessed and the results pre- oncomodulin levels seen in transformed rodent and sented in Table 1. The transformation frequency is not human cell lines in vitro (MacManus et al. 1982). linear with DNA concentration, since such a linear To characterize the influence of oncomodulin ex- relationship occurs only at low DNA concentrations pression on patterns of cell growth, the MTONCO2-7 (<20ng), and it has previously been shown that as the and MTONCO2-12 cell lines were assessed for their amount of DNA becomes saturating the specific trans- growth rate in culture, the ability of cells to form colonies Table 1. Effect of oncomodulin expression on the in soft agarose and the ability of cells to form dense foci at frequency of neoplastic transformation confluence. For comparison, the growth patterns of the Rat-1, NEO5 and Rat-ISMLT cell lines were also Transformation DNA concentrationf Cell line frequency* evaluated. Rat-ISMLT is a polyomavirus-transformed 0'S) Rat-1 cell line derivative that expresses all three Rat-1 2075 0.1 T-antigens, which was isolated following the introduc- 2195 0.05 3250 0.01 tion of the pSV2NEOSVEBla plasmid into the Rat-1 cell line. The histogram in Fig. 4 indicates the growth rate of NEO5 560 0.1 760 0.05 the various cell lines. Both the MTONCO2-7 and 1300 0.01 MTONCO2-12 have growth rates comparable to the parental and pSV2NEO-containing cell lines, while the MTONCO2-7 700 0.1 580 0.05 transformed Rat-ISMLT cell line displays a faster 750 0.01 growth rate and is capable of attaining higher cell MTONCO2-12 695 0.1 densities. In addition, the ability of oncomodulin-pro- 740 0.05 ducing cell lines to grow without solid support was 1900 0.01 determined. Cells were seeded in soft agarose and al- lowed to grow for a period of 7-10 days. As shown in * Calculated as the total number of foci per /ig of pSV2NEOSVEBla DNA. Fig. 5, only the polyomavirus-transformed cell line Rat- tConcentration of pSV2NEOSVEBla DNA per 100mm dish. ISMLT was able to grow without solid support, and

• Rat-1 63 Rat-1 SMLT 13 NE05 0 MTONCO2-7 Fig. 4. Histogram representating • MTONCO2-12 the growth rate of cell lines in culture. Included are the growth of the parental Rat-1 cell line, a pSV2NEOSVEBla-transformed cell line (Rat-lSMLt), a pSV2NEO transfected cell lines (NEO5), and two independently isolated MTONCO/pSV2NEO co- 3 5 7 transfected cell lines (MTONCO2- Number of days in culture 7 and MTONCO2-12).

Role of oncomodulin in transformation 521 Rat-1 MTONCO2-7

Fig. 5. Growth of cells in soft agarose. Only the polyomavirus-transformed cell line (Rat-ISMLT) was able to form colonies in a semi-solid environment. Neither the parental cell line (Rat-1) nor the oncomodulin-expressing cell lines (MTONCO2-7 and MTONCO2-12) displayed appreciable growth in soft agarose. Cells were photographed through a dissecting Rat-1 SMLT MTONCO2-12 microscope at a magnification of X15. forming activity declines until a plateau is reached (Mes Table 2. Effect of oncomodulin expression on the and Hassell, 1982). Nonethless, the expression of onco- ability to clone and propagate BMK cells in culture modulin does not appear to potentiate cellular transform- Cell lines established/ ation, as the MTONCO2-7 and MTONCO2-12 cell lines G418-resistant clones picked displayed approximately the same frequency of transformation after transfection with pSV2NEOSVEBla Transforming Experiment DNA as the NEO5 control. By comparing their rate of DNA* 1 2 growth in culture and their ability to form colonies in soft pSV2NEOSVEBla 4/6 2/3 5/9 agarose, pSV2NEOSVEB la-transformed derivatives of pSV2NEOSVEBla 2/2 1/3 6/9 both the MTONCO2-7 and MTONCO2-12 cell lines +pMTONCO PSV2NEO+pMTONCO 0/40 0/12 0/16 displayed growth patterns indistinguishable from pSV2NEO 0/40 pSV2NEOSVEBla-transformed Rat-1 cells. *DNA used to transfect BMK cells.

Expression ofoncomodulin in pnmary cells Some oncogenes, while unable to transform established plasmid could sustain continued growth in culture, while cell lines in culture, are capable of indefinitely prolonging no cell lines could be established after transfection of the lifespan of primary cells in culture and have sub- primary cells with pMTONCO DNA alone. sequently been referred to as immortalizing genes (Ras- To ensure that the oncomodulin mRNA could be soulzadegan et al. 1982; Land et al. 1983; Ruley, 1983). produced in primary cells, the pMTONCO and An example of this type of immortalizing gene is the large pSV2NEOSVEBla plasmids were co-transfected onto T-antigen of polyomavirus. In order to assess whether BMK cells and three G418-resistant cell lines were the expression of oncomodulin could confer an immortal established. RNA was extracted from these cell lines, phenotype on primary cells, 10 /ig of pMTONCO plas- separated by gel electrophoresis and probed for the mid was co-transfected with 10 ^g of pSV2NEO plasmid presence of oncomodulin-specific RNA sequences. The per 100 mm dish of BMK cells. As controls, both results are presented in Fig. 6. While all three cell lines pSV2NEO and pSV2NEOSVEBla were independently produced polyomavirus-specific RNA (data not in- transfected onto BMK cells at a concentration of 10/.ig of cluded), at least one cell line also expressed RNA specific plasmid DNA per 100 mm plate. In all cases cells were to oncomodulin. Thus, we conclude that the put under G418 selection and G418-resistant colonies pMTONCO construct is expressed in primary cells. were cloned. The survival rate of individual colonies is Since there appears to be no impediment to oncomodulin presented in Table 2. Greater than 50% of primary cells expression in primary cells, the inability to isolate cell isolated after transfection with the pSV2NEOSVEBla lines after transfection of the pMTONCO plasmid onto

522 A.-M. Mes-Masson et al. BCD and the MTONCO2-12 cell line utilizes the SV40 polyadenylation signal. It is not know why these different cell lines employ different polyadenylation signals, although it is clear that the SV40 signal is normally the preferred site, since four of the five oncomodulin RNA-producing cell lines utilize the SV40 polyadenylation signal (data not included). The pMTONCO plasmid did direct the synthesis of the oncomodulin protein, since it was shown -28S that only in the MTONCO2-7 and MTONCO2-12 cell lines could a protein of predicted molecular weight be specifically immunoprecipitated with antiserum directed against oncomodulin. Since the level of oncomodulin protein, as judged by radioimmuneassay, was comparable to the level seen in transformed cell lines (MacManus et al. 1982), we presume that the MTONCO2-7 and MTONCO2-12 cell lines are suitable candidates for the analysis of oncomodulin gene function. Two oncomodulin-producing cell lines, MTONCO2-7 and MTONCO2-12, were derived from Rat-1 cells, an established rat cell line. Both cell lines were compared with an oncogenically transformed cell line, since many of the phenotypic changes unique to transformed cells affect the rate and pattern of cell growth. The MTONCO2-7 and MTONCO2-12 cell lines showed similar density-dependent growth inhibition present in nontransformed cell lines (Holley and Kiernan, 1968). Fig. 6. Northern blot analysis of G418-selected By contrast with normal cells, many transformed cell pSV2NE0SVEBla/MT0NC0 co-transfected primary BMK lines grow efficiently in semi-solid medium (MacPherson cells. The arrow by lane D indicates the position at which and Stoker, 1964). Oncomodulin-producing cell lines oncomodulin RNA migrates within a 1.0% agarose/ were unable to form colonies in soft agar, and displayed formaldehyde gel. Of RNA extracted from three randomly growth rates and saturation densities comparable to the selected cell lines (lanes B, C and D) producing polyomavirus-specific RNAs, at least one cell line (lane C) untransformed parental cell line. Therefore, by a number also produces oncomodulin-specific RNA from integrated of criteria, the presence of oncomodulin in an established MTONCO sequences. cell line does not appear to confer growth characteristics normally associated with cellular transformation. We have not tested whether the MTONCO2-7 and BMK cells indicates that oncomodulin is not an immorta- MTONCO2-12 cell lines are able to form tumors in lizing agent in vitro. syngeneic animals. However, since a loose correlation exists between growth in soft agarose in vitro and tumor Discussion formation in vivo (Freedman and Shin, 1974; Shin et al. 1975), oncomodulin-producing cells would not be As rat oncomodulin is expressed in a wide variety of expected to be tumorigenic, although this possibility tumors, it was of interest to determine if oncomodulin in cannot be dismissed entirely. any way contributes to or facilitates the development of To investigate whether oncomodulin might act in a cancer. In this initial study, it was investigated whether more subtle manner to influence oncogenic transform- oncomodulin acts in a manner analogous to oncogenes in ation, the MTONCO2-7 and MTONCO2-12 cell lines vitro. For this purpose, an expression vector containing were transformed with the pSV2SVEBla plasmid. While the rat oncomodulin cDNA was introduced into both an oncomodulin is not able to initiate transformation of Rat- established cell line and primary cells derived from 1 cells, it is conceivable that the presence of oncomodulin mouse kidney. might make cells more susceptible to the effects of a A recombinant plasmid (pMTONCO), in which the transforming oncogene. From our results we conclude synthesis of oncomodulin-specific RNA is under the that, in this in vitro system, expression of oncomodulin direction of the metallothionein promotor, was used in does not increase the transforming frequency of the these studies. The pMTONCO plasmid was introduced pSV2NEOSVEBla plasmid, nor does it appear to affect into Rat-1 cells and the expression of oncomodulin- the growth characteristics of polyomavirus-transformed specific RNA was verified. The two cell lines chosen for cells. further study, MTONCO2-7 and MTONCO2-12, pro- Since the highest expression of oncomodulin is nor- duced oncomodulin-specific RNA of different length. We mally found in the outer placenta (Brewer and believe this difference is due to differential utilization of MacManus, 1985), it is tempting to speculate that polyadenylation signals, where MTONCO2-7 cell line oncomodulin might function to enhance tumor invasive- uses the authentic oncomodulin polyadenylation signal, ness or metastasis. This hypothesis seems unlikely, since

Role of oncomodulin in transfonnation 523 oncomodulin is also normally present in non-invasive acid from sources enriched in ribonucleases. Biochemistry 18, amnion (Brewer and MacManus, 1985), and it has been 5294-5299. shown that no correlation exists between the level of DURKIN, J. P., BREWER, M. L. AND MACMANUS, J. P. (1983). Occurence of the tumor-specific, calcium-binding protein, oncomodulin expression and tumor vigor (MacManus oncomodulin, in virally transformed normal rat kidney cells. and Whitfield, 1983). Oncomodulin-producing polyoma- Cancer Res. 43, 5390-5394. virus-transformed cells were not tested for their ability to FREEDMAN, V. AND SHIN, S. (1974). Cellular tumorigenicity in nude form tumors in animals, although this analysis would mice: Correlation with cell growth in semi-solid medium. Cell 3, directly address whether the expression of oncomodulin 355-359. FREEMAN, A. E., GILDEN, R. V., VERNON, M. L., WOLFORD, R. G., confers a growth advantage to transformed cells in vivo HUGUNIN, P. E. AND HUEBNER, R. J. (1973). Bromo-2'- during tumor formation. deoxyundine potentiation of transformation of rat-embryo cells Some oncogenes, while not able to transform cells in induced in vitro by 3-methylcholanthrene: Induction of rat culture, appear to be able to indefinitely prolong the leukemia virus gs antigen in transformed cells. Proc. natn. Acad. Sci. U.S.A. 70, 2415-2419. lifespan of primary cells in cultures and have been FRESHNEY, R. I. (1987). Culture of Animal Cells: A Manual of Basic referred to as immortalizing genes (Rassoulzadegan et al. Technique. Alan R. Liss, Inc., New York. 1982; Land et al. 1983; Ruley, 1983). Though we have GILLEN, M. F., BANVILLE, D., RUTLEDGE, R. G., NARANG, S., demonstrated that oncomodulin-specific RNA can be SELIGY, V. L., WHITFIELD, J. F. AND MACMANUS, J. P. (1987). A produced by the pMTONCO construct in primary cells, complete complementary DNA for the oncodevelopmental calcium- binding protein, oncomodulin. J'. biol. Chem. 262, 5308-5312. we were unable to show that expression of oncomodulin HOLLEY, R. AND KIERNAN, J. (1968). "Contact inhibition" of cell altered the lifespan of primary cells in culture. Thus we division in 3T3 cells. Pivc. natn. Acad. Sci. U.S.A. 60, 300-304. conclude that oncomodulin does not act in a manner KLEE, C. B. AND HEPPEL, L. A. (1984). The effect of oncomodulin similar to immortalizing agents. on cAMP phosphodiesterase activity. Biochem. biophys. Res. Commun. 125, 420-424. To date, our attempts to uncover an oncomodulin- LAEMMLI, U. K. (1970). Cleavage of structural proteins during the specific activity that would play a role in tumor develop- assembly of the head of bacteriophage T4. Nature, Ijond. Zll, ment have been unsuccessful. While the results presented 680-684. here rely heavily on in vitro tissue culture systems, LAND, H., PARADA, L. F. AND WEINBERG, R. A. (1983). Tumorigenic conversion of primary embryo fibroblasts requires at parallel experiments were initiated to assess the role of least two cooperating oncogenes. Nature, Land. 304, 596-601. oncomodulin in vivo. For this purpose the pMTONCO MACMANUS, J. P. (1979). Occurrence of a low-molecular weight plasmid was microinjected into fertilized mouse embryos calcium-binding protein in neoplastic liver. Cancer Res. 39, in order to generate transgenic mice. The results of these 3000-3005. experiments will be discussed extensively elsewhere MACMANUS, J. P. (1981a). Development and use of quantitative immunoassay for the calcium-binding protein (molecular weight, (Chalifour et al. 1989). However, it appears that ex- 11,500) of Morris 5123. Cancer Res. 41, 974-979. pression of oncomodulin is incompatible with the normal MACMANUS, J. P. (19816). The stimulation of cyclic nucleotide development of the mouse, and live births of oncomodu- phosphodiesterase by an Mr 11,500 calcium-binding protein from lin-expressing transgenic animals were not observed. rat hepatoma. FEBS Lett. 126, 245-249. Though we can conclude that oncomodulin does not act MACMANUS, J. P. AND WHITFIELD, J. F. (1983). Oncomodulin - A calcium-binding protein from hepatoma. In Calcium and Cell like an oncogene in vitro, we cannot rule out the Function (ed. W. Y. Cheung), pp. 411-440, vol. 2. Academic possibility that the expression of oncomodulin in some Press, New York. way influences tumor formation in vivo. MACMANUS, J. P., WATSON, D. C. AND YAGUCHI, M. (1983). The complete ammo acid sequence of oncomodulin - a parvalbumin- We thank J. A. Hassell, J. P. MacManus and B. Massie for like calcium-binding protein from Morris hepatoma 5123tc. Eur.J. providing some of the reagents used in these studies. We are Biochem. 136, 9-17. MACMANUS, J. P., WHITFIELD, J. F. AND STEWART, D. J. (1984). grateful to J. P. MacManus for performing the radioimmune- The presence in human tumours of a M, 11,700 calcium-binding assays. We also thank F. Gossard for helpful discussions and protein similar to rodent oncomodulin. Cancer Ijett. 21, 309-315. critical reading of the manuscript. MACMANUS, J. P., WHITFIELD, J. F., BOYNTON, A. L., DURKIN, J. P. AND SWIERANGA, S. H. H. (1982). Oncomodulin - A widely distributed, tumour-specific calcium-binding protein Oncodevl Biol. Med. 3, 79-90. References MACPHERSON, I. AND STOKER, M. (1964). Agar suspension culture for the selective assay of cells transformed by polvoma virus. BOYNTON, A. L., MACMANUS, J. P. AND WHITFIELD, J. F. (1982). Virology U, 147-ISl'. Stimulation of liver cell DNA synthesis by oncomodulin, an MW MANIATIS, T., FRITSCH, E. F. AND SAMBROOK, J. (1982). Molecular 11,5000 calcium-binding protein from rat hepatoma. Expl Cell Res. Cloning -A Laboratory Manual. Cold Spring Harbor Laboratory 138, 454-458. Press, Cold Spring Harbor, New York. BREWER, L. M. AND MACMANUS, J. P. (1985). Localization and MES, A.-M. AND HASSELL, J. A. (1982). Polyoma viral middle T synthesis of the tumor protein oncomodulin in extraembryonic antigen is required for transformation. J'. Virol. 42, 621-629. tissues of the fetal rat. Devi Biol. 112, 49-58. MULLER, W. J., NAUJOKAS, M. A. AND HASSELL, J. A. (1984). CHALIFOUR, L. E., GOMES, M. L. AND MES-MASSON, A. M. (1989). Isolation of large T antigen-producing mouse cell lines capable of Microinjection of metallothionein-oncomoduhn DNA into fertilized supporting replication of polyomavirus-plasmid recombinants. mouse embryos is correlated with fetal lethality. Oncogene 4, in Molec. cell. Biol. 4, 2406-2412. press. MUTUS, B., KARUPPIAH, N., SHARMA, R. K. AND MACMANUS, J. P. CHALIFOUR, L. E., WIRAK, D. O., WASSARMAN, P. M. AND (1985). The differential stimulation of brain and heart cyclic-AMP DEPAMPHILIS, M. L. (1986). Expression of Simian Virus 40 early phosphodiesterase by oncomodulin. Biochem. biophvs. Res. and late genes in mouse oocytes and embryos. J. Virol. 49, Commun. 131, 500-506. 619-627. PFYFFER, G. E., HAENNERLI, G. AND HEIZMANN, C. W. (1984). CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J. AND Calcium-binding proteins in human carcinoma cell lines. Proc. RUTTER, \V. J. (1979). Isolation of biologically active ribonucleic natn. Acad. Sci. U.S.A. 81, 6632-663.

524 A.-M. Mes-Masson et al. RASSOULZADECAN, M., COWIE, A., CARR, A., GLAICHENHAUS, N., SOUTHERN, P. J. AND BERG, P. (1982). Transformation of KAMEN, R. AND CUZIN, F. (1982). The roles of individual mammalian cells to antibiotic resistance with a bacterial gene under polyoma virus early proteins in oncogenic transformation. Nature, control of the SV40 early region promoter, jf. molec. appl. Genet. Umd. 300, 713-718. 4,327-341. RULEY, H. E. (1983). Adenovirus early region 1A enables viral and WIGLER, M., PELLICER, A., SILVERSTEIN, S. AND AXEL, R. (1978). cellular transforming genes to transform primary cells in culture. Biochemical transfer of single-copy eucaryotic genes using total Nature, Loud. 304, 602-604. cellular DNA as donor. Cell 14, 725-731. SHIN, S., FREEDMAN, V, RISSER, R. AND POLLACK, R. (1975). Tumongenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. natn. Acad. Sci. U.S.A. 72, 4435-4439. (Received 8 May 1989 - Accepted 25 July 1989)

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