transformation of nih/3t3 cells by ornithine decarboxylase ......jeffrey a. moshier,2 julie dosescu,...

(CANCER RESEARCH 53. 2618-2622. June I. 1993]

Transformation of NIH/3T3 Cells by Ornithine Decarboxylase Overexpression1

Jeffrey A. Moshier,2 Julie Dosescu, Magdalena Skunca, and Gordon D. Luk

Departments nf Internal Medicine IJ. A. M.. J. D., M. S.I Â¡milMolecular Biology and Genetics Â¡J.A. M.Â¡.Wayne State University Sellimi of Medicine. Detroit, Michigan 48201.tnul Dalias VA Medical Cenlcr and University of Texas Southwestern Medicai Center Â¡G.D. LI. Dallas. Texas 75216

ABSTRACT

Ornithine decarboxylase (ODO plays a rate-limiting role in polyamine

biosynthesis and is intimately associated with cell proliferation and function. Although elevated levels of ODC mRNA, protein, and enzyme activityare consistently detected in transformed cells and tumors, the questionremains as to whether ODC gene overexpression has a causative role intumorigenesis. We have stably transfected MH/3T3 fibroblasts with anexpression construct containing human ODC complementary DNA underIranscriptional control of the human ÃŸ-actinpromoter. Cells transfectedwith the ÃŸ-actin/ODC DNA construct, designated NODI cells, and control

transfectants, termed M K cells, were analyzed for ODC gene expressionand cell growth characteristics. ODC activity and mRNA levels wereelevated 3-6-fold in NODC cells relative to M K cells. MODC cells, incontrast to M K control cells, are not contact inhibited, exhibit anchorage-

independent growth, cycle more rapidly, and induce tumors in nude micemore efficiently and rapidly. These results directly establish a causativerole for the misregulation of ODC gene expression in the acquisition of atransformation phenotype and provide a model to examine the interactionof ODC and other gene products in neoplastic development.

INTRODUCTION

The accumulation of genetic alterations during tumorigenesis has aprofound impact on the deregulation of cell proliferation. Defining theeffect of a specific alteration in gene function on neoplastic development is complicated by the myriad attendant changes in cell metabolism, ultrastructure, and proliferative control. ODC.1 a key regulatory

enzyme in polyamine biosynthesis, is an example of a highly regulated, proliferation-related enzyme for which a causative role in neo

plastic transformation has been intimated but not directly established(reviewed in Refs. 1-4). ODC has been shown to be crucial to cell

growth by experimental approaches that interfere with the function ofthe enzyme. For example, inhibition of ODC activity by the specificinhibitor difluoromethylornithine arrests the growth of cells in cultureand animal tumors in vivo (5). In addition, a mutant Chinese hamsterovary cell line lacking endogenous ODC activity does not proliferatein culture unless supplied with exogenous polyamines (6) or transfected with a functional ODC gene (7).

Several lines of evidence indicate that aberrations in ODC regulation, and subsequent polyamine accumulation, are intimately associated with neoplastic transformation: (a) elevated levels of ODC geneproducts are consistently detected in transformed cell lines (8), virtually all animal tumors (5. 9), and in certain tissues predisposed totumorigenesis (10-12). Perhaps the best example of this correlation is

the stepwise increase in ODC activity observed during the progressionof normal colon mucosa to benign colon polyp and finally to colonadenocarcinoma (11. 13. 14); (b) the induction of ODC activity andpolyamine accumulation is essential for carcinogenesis in a number ofexperimental animal models such as 12-O-tetradecanoylphorbol-13-

Received 12/8/92; accepted 3/26/9Ã•.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grant CA-51206 from the National Cancer Institute andby Grant IN-162 from the American Cancer Society.

2 To whom requests for reprints should he addressed."The abbreviations used are: ODC. ornilhine decarboxylase (EC 4.1.1.17): DMEM.

Dulbecco's modified Eagle's media: cDNA. complementary DNA: FBS. fetal bovine

serum.

acetate promotion of mouse skin tumors (10). 1,2-dimethylhydrazineinduction of mouse colon tumors (15). 1-methyl-l-nitrosourea induc

tion of rat mammary tumors (16. 17), and heterotopically transplantedrat bladder tumors (18); (c) overexpression of ODC in variant tumorcell lines is associated with the acquisition of a growth phenotypecharacteristic of transformed cells. Specifically, difluoromethylorni-thine-resistant tumor cells selected for ODC overexpression exhibit anincreased proclivity for anchorage-independent growth (19); (d) on

cogene transformation of cells in culture is accompanied by alterationsin ODC and polyamine biosynthesis. For example, rodent fibroblaststransfected with an activated ras oncogene, but not with a normal rasgene, displayed enhanced ODC activity and a transformed phenotype(20). As compelling as these correlations may be. they do not provethat a causative relationship exists between abnormal ODC regulationand tumorigenesis. In each case, the observed phenotypic changesmay be due to accompanying alterations in other growth-regulating

functions.In this study, we have developed derivatives of NIH/3T3 cells that

stably express elevated levels of ODC under the control of the humanÃŸ-actingene promoter. Various growth characteristics of these cells

are compared to transfected control cells which differ initially only inODC gene regulation. Cells expressing ODC from the heterologouspromoter exhibited proliferation properties characteristic of transformed cells such as loss of contact inhibition, efficient anchorage-

independent cell growth, and increased tumorigenicity. These resultssubstantiate the role of ODC in cell proliferation and neoplastic transformation.

MATERIALS AND METHODS

ODC Expression Vector. Plasmici construct pÃŸ-ODCwas designed to express human ODC under the control of the human ÃŸ-actinpromoter. A 1768-

base pair Hind\\\ restriction fragment containing human ODC cDNA and 29base pairs of pBR322 vector was excised from cloned DNA pODC2H/10.provided by Dr. O. A. JÃ¼nni(University of Helsinki. Helsinki. Finland) (21).and ligaled into the Hiiul\\\ site of plasmid pLK-444 (also called pHÃŸAPr-l-

neo) (22). contributed by Dr. L. Kedes (Stanford University). The ODC cDNAinsert in pla.smid pÃŸ-ODClacks the first 248 leader sequences hut includes theentire protein-coding region in the appropriate orientation to express ODCmRNA from the ÃŸ-actinpromoter (Fig. 1). Standard molecular biology pro

cedures were performed according to protocols described by Ausubel et ai.(23).

Cell Culture and Transfection Conditions. NIH/3T3 cells (AmericanType Culture Collection) maintained in DMEM with high glucose and I09rFBS were stably transfected by the calcium phosphate protocol of Gorman(24). Briefly. NIH/3T3 cells were subcultured and reseeded at a density of 9 x10s cells/75-cnr flask. The cells were transfected the next day with 10 (jg/flask

of pÃŸ-ODCor pLK-444 DNA using a CellPhect transfection kit (Pharmacia.Piscataway. NJ) according to the manufacturer's instructions. After 6 h the

cells were treated for 1 min with 15% glycerol in 4-(-2-hydroxyethyl)-l-pipera/ineethanesulfonic acid-buffered saline (pH 7.12) at room temperature,

washed, and returned to culture medium to recover. Cells were subsequentlyharvested and reseeded at a concentration of I x IO6 cells/100-mm2 plate in

10 ml of selection medium containing 4(K) )jg/ml G418 (Geneticin; BethesdaResearch Laboratories. Gaithersburg. MD). Surviving cells were refed withselection media and dead cells were removed every 3 days until day 13.G418-resistant colonies were isolated with cloning cylinders and grown inDMEM with high glucose and \0<7cFBS. Transfected cells are rechallenged

periodically with G4I8 to maintain the phenotype.

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NIH/.Vn IRANSFORMATION BY ORNITHINE DECARBOXYLASF:

ODC Enzyme Assay. ODC activity was determined using the method ofBeaven et al. (25). Protein concentration was determined by the method ofBradford using a kit from Bio-Rad according to the manufacturer's instruc

tions.Northern Blot Analyses. Total RNA was isolated from logarithmically

growing cell cultures according to the protocol of Cathala et al. (26). RNAsamples (10 ug) were size-fractionated by electrophoresis in 1.5% agarose

gels containing 6% formaldehyde and 20 rmi sodium phosphate (pH 6.8).stained with ethidium bromide, transferred to Gene Screen (New EnglandNuclear, Boston. MA) nylon membranes by capillary blotting, and baked at80Â°Cfor 2 h. A gel-purified 1.8-kilobase froRI fragment from pODC2H/l()was radiolabeled (27) to an average specific activity of 5 x 10" cpm/ug with[a-'-PJdCTP (New England Nuclear) using a random primers DNA-labeling

kit (Pharmacia). RNA filters were prehybridi/.ed in 50% formamide. 0.2%polyvinylpyrrolidone. 0.2% Ficoll. 0.2% bovine serum albumin. 0.05 M Tris-

HCI (pH 7.5), 1.0 M NaCl, 0.1% sodium pyrophosphate, 1% sodium dodecylsulfate. 10% dextran. and 100 ug/ml of denatured salmon sperm DNA at42Â°C.Hybridi/ations were performed for 16 h at 42Â°Cin the same solution

except that NaCl concentration was lowered to 0.75 M and radioactive probewas included. The final washing of the membranes was performed at 55Â°Cin

75 HIMNaCl and 7.5 ITIMsodium citrate containing 0.5% sodium pyrophos-phate. Membranes were exposed to X-Omat film with Cronex intensifying

screens. The resultant hybridization signals were quantitated from the membranes using an Ambis Radioanalytical Imaging System (AMBIS Systems.Inc., San Diego, CA). The intensity of each signal was normalized to I8SrRNA which was quantitated by reprobing the membranes with radiolabeled18S ribosomal DNA.

Anchorage-independent Cloning Assay. Cells were seeded at a concentration of 1 x IO' cells/35-mm plate in DMEM supplemented with 18% FBS.

6.7 ITIMsodium bicarbonate. 1.25 imi sodium pyruvate. 0.05 msi essential andnonessential amino acids, 2 imi glutamine. 0.1 imi serine. 0.15 imi asparagine.120 units/ml penicillin, and 120 ug/ml streptomycin (GIBCO. Grand Island.NY) all gelled with 0.3% agarose (SeaPlaque: FMC. Rockland. ME) over a0.99}-agarose underlayer containing 2.5 ml of conditioned media and 4.5 ml ofthe above DMEM-enriched media.

Tumorigenicity Assay in Nude Mice. Cells were isolated from monolayerculture, washed, and resuspended in phosphate-buffered saline. Athymic nudemice (CD-I nu/nu; Charles River Laboratories. Wilmington, MA) were inoculated s.c. in both Hanks with 5 x 10" cells/flank. Tumor sizes and animal

weights were measured weekly and tumors were harvested before they reached2 cm in diameter. Tumor samples were fixed in Bouin-Hollande's solution.

sectioned, and stained with hematoxylin-eosin for pathological examination.

RESULTS

To directly assess the effects of ODC gene overexpression on cellgrowth properties, we constructed expression plasmid pÃŸ-ODCbyplacing the full coding region of human ODC cDNA under the tran-scriptional control of the human ÃŸ-actingene promoter (Fig. 1). Pre-

5'UTR IVS1

HumanÃŸ-actinPromoter

SV-poly A

Ampr

Fig. I. Expression vector pÃŸ-ODC.Human ODC cDNA (Ãœ)was inserted into expression vector pLK-444 downstream of the human ÃŸ-actinpromoter (03 ' untranslated region(5'UTR; â€¢¿�land first intervening sequence (IVSI: E3j. The ODC 3' terminus is hounded

by an SV40 polyadenylation signal SV-poly A; Q. The vector contains ampicillin- andneomycin-resistant genes for selection in prokaryotic and cukaryotic cells, respectively.

50% confluence

100% confluenceNLK-2 NLK-3 NODC-1 NOOC-2 NODC-5 NODC-6

Fig. 2. ODC activities in transfected NIH/3T3 cells. A representative assay of ODCactivities in NODC cells transt'ected with pÃŸ-ODCexpression construct and NLK control

cells transfected with the vector DNA is shown. Exponentially growing (B) and confluentcells (D) were assayed as described in "Material and Methods." ODC activity is expressed

in pmol of COi released from [ l4C|ornithine/h/mg of protein.

vious experiments have demonstrated that the parental expressionvector pLK-444 (also called pHÃŸAPr-1-neo) containing the humanÃŸ-actinpromoter directs high-level gene expression in a wide variety

of cells including NIH/3T3 fibroblasts (22). Both plasmids contain theaminoglycoside phosphotransferase gene, which confers resistance tothe antibiotic G418 under the control of the SV40 early promoter.

Plasmids pÃŸ-ODCand pLK-444 were stably transfected into NIH/

3T3 cell and transfected cells were selected with G4I8. After 2 weeksin selective medium, there were 54 G418-resistant colonies Â¡npÃŸ-ODC-transfected cells and 47 in pLK-444-transfected cells. The trans-fection frequencies representing the number of G418-resistant colonies per IO6 cells plated were 13.5 and 11.75 for pÃŸ-ODCand pLK-

444-transfected cells, respectively. All G4l8-resistant coloniestransfected with pÃŸ-ODCexhibited loss of contact inhibition and

formed foci at high cell densities. The morphology of these cellschanged from a spindle shape to a more rounded contour at increasingconfluence. In contrast, the pLK-444-transfected colonies grew ascontact-inhibited monolayers and displayed no morphological alter

ations. Representative colonies were isolated from both transfectedcell types and designated NODC (NIH/3T3 cells transfected withpÃŸ-ODC)and NLK (NIH 3T3 cells transfected with pLK-444).

The level of ODC enzyme activity present in NODC and NLK cellswas determined in cells at log phase growth and at confluency (Fig.2).4 Growing NODC cells consistently exhibit levels of ODC activity

ranging 3-6-fold greater than levels detected in NLK cells. The average level of ODC activity in NODC cells is 4.0-fold greater than that

of NLK cells. At 100% cell confluence, the ODC activity of NODCcells, which was 4-11-fold lower than at 50% confluence, was still

comparable to that of exponentially growing NLK control cells. Thedrop in ODC activity of confluent NLK cells is comparable to that ofresting fibroblasts (data not shown).

The difference in ODC gene expression between proliferatingNODC and NLK cells is also reflected in ODC mRNA levels (Fig. 3).Direct quantitation of ODC mRNA detected by Northern blot analysesreveals a 2.8-5-fold increase in ODC mRNA in NODC cells com

pared to NLK cells. The human ODC cDNA probe is 85% homologous to mouse ODC transcripts; therefore under the conditions used(Fig. 3) both endogenous and human ODC mRNAs are detected.

4 fnpuhlished data.

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NIH/3T3 TRANSFORMATION BY ORNITHINF. OF.CARBOXYt.ASE

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OOO

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18SFig. 3. Northern blot analysis of ODC mRNA in transfected NIH/3T3 cells. Total RNA

from NODC and NLK cells was isolated, fraciionaied hy electrophoresis in a denaturingagarose gel. transferred to a nylon membrane, and hybridized to '-P-rudiolaheled humanODC cDNA (/o/i) as described in "Materials and Methods." The tiller was stripped and

reprobed with IKS ribosomal DNA to normali/e lor variation in sample loading betweenlanes {hoilomi. Quantitation of signal intensities was performed hy direct scanning of theNorthern blot.

Table I Chanclertala nf NIH/JTi cells tmnsfecled with Of)C espressimi vfcinrTumors/inoculum'1

ContactCelllineNLK-2NLK-3NODC-INODC-2NODC-5NODC-6inhibitionlime(hl+

18.2+21.6ND'14.412.0NDefficiency

(*)0.050.10ND5338ND4wk0/140/106/616/1616/166/620wk3/14l/Kl6/616/1616/166/6

" One x 10* cells were plated in 0.3ri agarose with a 0.9^ agarose underlayer.'' Five x 10" cells were injected s.c. per mouse Hank. Shown are the number of tumors

detected 4 and 20 weeks poslinoculation.' ND. not determined.

The influence of ODC gene overexpression on NIH/3T3 cells wasalso examined by monitoring the acquisition of transformation phe-

notypes. Table I summuri/.es the growth properties of the transtectedNIH/3T3 cells. As noted, all NODC cell lines have lost the ability tobe growth inhibited by cell contact. Fig. 4 shows a representative

example of a multilayered colony formed by overgrown NODC cells.In addition, NODC cells exhibited doubling times which were 33%shorter than those of NLK cells (Table 1).

We also examined the transfectants for anchorage independentgrowth in culture (Fig. 5). The colony-forming efficiency of NODCcells in agar was in the 40-50% range (Table I ) and comparable tothose of long terminal repeat-driven v-H-ra.v and v-i-r/MransformedNIH/3T3 (28). In contrast, NLK cells displayed no anchorage-inde

pendent growth. Moreover, each NODC cell line induced tumorswithin 4 weeks in all mice inoculated. NLK cells, in contrast, did notproduce tumors within 8 weeks alter injection of 5 X 10'' cells/flank.

One mouse given an injection of NLK-2 cells developed a small

nodule at 10 weeks postinjection which grew to 1.2 cm by 5 months.Three other sickly mice developed small tumors (<0.6 cm) at 20weeks.

DISCUSSION

In this study, we describe the tumorigenic conversion of NIH/3T3cells following transfection with a human ODC cDNA under tran-scriptionul control of a human ÃŸ-actingene promoter. Cells trans

fected with the ODC expression construct display elevated constitutive levels of ODC activity and mRNA levels compared to controltransfectants. ODC gene regulation in these cells may also be alteredby the absence of noncoding 5'-flanking sequences involved in trans-

lational control of ODC (29-31) and/or by temporal misregulation of

ODC expression due to the heterologous promoter. In any event, themisregulation of ODC expression in transfected NIH/3T3 cells produced significant changes in growth properties such as decreasedpopulation-doubling time, loss of contact inhibition, anchorage-inde

pendent growth and colony formation, and tumor induction in nudemice. These results directly substantiate the importance of ODC to cellproliferation and establish a causative role for ODC gene misregulation in the acquisition of transformation phenotypes.

The overproduction of ODC is thought to confer a growth advantage to mammalian cells. Supporting evidence for this suppositioncomes from studies reporting increased growth potential of culturedcells overexpressing endogenous ODC. For example, mouse L12IOand myeloma cells selected for resistance to ODC-inhibiting drugs

Fig. 4. Loss of contact inhibition hy transfectedNIH/3T3 cells overexpressing ODC. RepresentativeG4IS-resistant colonies derived from NLK controlcells ilfft) and NODC cells overexpressing ODC(rifilili were grown to confluency. Despite overcrowding, the NLK cells remain a monolayer at saturation densities. NODC cells, in contrast, show lossof cell-to-cell contact inhibition hy forming multilay-

ered foci.

; ,'. Ã•** . ' "â€¢¿� " V

l

2620



NIH/.m TRANSFORMATION BY ORNITHINE DECARBOXYLASE

â€¢¿�â€¢¿�

*

Ã¨<t

NLK-2 NODC-2 NODC 5Fig. 5. Anchorage-independent growth of NIH/.TT3 Iransfectants overexpressing ODC. One x 10' NLK-3 control cells (left] and NODC-2 and NODC-3 cells overexpressing ODC

(middle and righi, respectivelyl were grown in 0.3% agarose with a 0.9% agarose underlayer. Colonies greater than 20 cells were scored and the efficiency of colony formation wasdetermined (Table I ).

contain elevated ODC levels relative to parental cells and grow muchmore efficiently in soft agar ( 19). Likewise, an Ehrlich ascites carcinoma cell line selected to overproduce ODC grew much more aggressively when inoculated into nude mice than its nonselected counterpart (32). In each of these cases, the cell lines were derived fromtumorigenic cells in which alterations in ODC regulation andpolyamine metabolism as well as other growth control pathways hadalready occurred. In addition, the processes used to select ODC overproducing cells could also introduce other unknown alterations. Ourresults concur and strengthen the hypothesis that ODC overproductiongives cells a growth advantage. In our study, nontumorigenic NIH/3T3cells acquired increased growth potential including growth in semi-

solid medium and in nude mice apparently by changes only in ODCproduction.

Recently, Hibshoosh et al. (33) overexpressed a construct containing mouse ODC in NIH/3T3 cells using a retrovirus-derived expression vector system. Infected cells overexpressing ODC 5â€”10-fold

showed no changes in morphological or growth properties. There aretechnical differences which may explain the discrepancies betweentheir results and ours. The major difference is that the expressionvector of Hibshoosh et al. synthesized a 5-kilobase RNA which included ODC and non-ODC sequences. It is not clear whether the ODC

protein itself was fused to a viral peptide. This could have an impacton the metabolism, localization, and control of the enzyme intracel-

lularly. Additional differences include the species from which theODC cDNA and promoters were derived. We utilized a human ÃŸ-actin

promoter, while Hibshoosh et al. used a Moloney murine leukemiavirus long terminal repeat. Thus, the conflicting results may be secondary to potential differences in expression throughout the cell cycleof the transfected ODC cDNA as directed by these two heterologouspromoters. A trivial explanation such as subtle changes in the NIH/3T3 cell lines maintained in the two laboratories may also account forthe discrepancy between the two reports.

Several lines of evidence indicate that ODC overexpression is notsufficient for malignant transformation of normal diploid cells. Incontrast to NIH/3T3 cells, transfection of embryo-derived rat fibro-

blast cell lines with ODC expression constructs does not dramatically

alter the growth of these cells (33).' The different effect of ODC

overexpression on these cell lines is most likely due to the differentstages of tumorigenic progression represented by each. NIH/3T3 cells,but not the rat fibroblast lines, are extremely sensitive to tumorigenicconversion by single oncogenes (34) and are susceptible to spontaneous transformation (35) as shown in these studies. Alterations duringthe establishment of NIH/3T3 cells in culture are consistent withadvanced stages of carcinogenesis and apparently contribute to ODC-

induced growth enhancement.More directly, transgenic mice that aberrantly express a human

ODC gene (36. 37) have not. to date, been reported to develop moretumors than controls. Interestingly, almost all tissues in the ODCtransgenic mice compensate for the elevated ODC activity in such away(s) (hat changes in cellular polyamine content are minimal. It isprobable that other genetic lesions affecting polyamine metabolismand/or other growth-regulating mechanisms are required to supple

ment ODC overexpression for tumorigenesis to occur in vivo. Candidate genes in which mutations are known to influence polyaminemetabolism and which may act cooperatively with ODC overexpression to drive tumorigenesis include oncogenes such as rax and mvc. Ithas been established that transformation by both activated rax and wvconcogenes is tightly coupled to ODC gene expression and polyamineaccumulation (20, 38. 39). For example, the growth rate of ras-

transformed NIH/3T3 cells is correlated with the level of ODC activity (40) and morphological transformation of rat fibroblasts is significantly increased in cells engineered to overexpress ODC by directtransfection (33). The consequence of oncogene activation onpolyamine metabolism may be oncogene specific as exemplified bythe observation that N-wyr-transfected rat fibroblasts do not exhibit

elevated ODC activity but rather accumulate polyamines by increasing polyamine transport into the cells (41). It is not unreasonable topostulate that normal cells may be able to compensate for a limitednumber of alterations in polyamine metabolism, such as elevatedODC. However, as lesions in growth-regulating genes accumulate incells undergoing neoplastic transformation, the ability to coumerbal-

' J. A. Mushier, unpublished observation.

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NIH/.Vn TRANSFORMATION BY ORNITHINE DECARBOXYLASK

ance alterations in ODC and polyumine regulation may be lost, leading to enhanced growth as demonstrated in transfected NIH/3T3 cellsin this study.

The transfected NIH/3T3 cell lines presented here provide a modelsystem to examine the involvement of other proliferation-associated

gene products that respond to misregulated ODC gene expression andwhich, in turn, contribute to subsequent processes implicated in celltransformation.

ADDENDUM

Auvinen el al. (42) recently reported similar findings on the effect of ODCoverexpression on NIH/3T3 cells transfected with human ODC cDNA.

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1993;53:2618-2622. Cancer Res Jeffrey A. Moshier, Julie Dosescu, Magdalena Skunca, et al. OverexpressionTransformation of NIH/3T3 Cells by Ornithine Decarboxylase

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transformation of nih/3t3 cells by ornithine decarboxylase ......jeffrey a. moshier,2 julie dosescu,...

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