Proc. Natl. Acad. Sci. USAVol. 84, pp. 1263-1267, March 1987Cell Biology

Alteration of the tumorigenic and metastatic properties ofneoplastic cells is associated with the process of calciumphosphate-mediated DNA transfection

(tumor progression/mutagenesis/gene expression/metastasis/genetic instability)

ROBERT S. KERBEL*t, CAROL WAGHORNE*, M. S. MAN*, BRUCE ELLIOTTt, AND MARTIN L. BREITMAN**Division of Cancer and Cell Biology, Mt. Sinai Hospital Research Institute, 600 University Avenue, Toronto, ON, Canada M5G 1X5, and the Department ofMedical Genetics, University of Toronto; and tDepartment of Pathology, Queen's University, Botterell Hall, Kingston, ON, Canada K7L 3N6

Communicated by Harry Rubin, October 17, 1986 (received for review July 24, 1986)

ABSTRACT During the course of studies designed toassess the effect of human Ha-ras gene expression on themalignant behavior of transfected mouse tumor cells we notedthat the process of Ca3(PO4)2-mediated DNA transfection wasitself associated with profound alterations in tumorigenic ormetastatic behavior. The cell line used as a recipient for thesestudies was a tumorigenic nonmetastatic CBA/J mouse mam-mary adenocarcinoma line called SP1. When cotransfectedwith plasmids containing the neo gene (pSV2neo) and theactivated Ha-ras gene (pT24-c3), cells from the pooled (5-10colonies) G418-resistant colonies gave rise to spontaneous lungmetastases in 85% of mice after subcutaneous inoculation.However, we noted that 17% of control mice inoculated withG418-resistant pSV2neo-transfected SP1 cells also had lungmetastases and that this number approached 100% as theinoculum comprised a greater pool size (50-100 colonies).When cell lines established from isolated pSV2neo-transfectedcolonies were examined, 3/16 were found to be metastatic. Wealso found that 3/16 clones grew slowly, or not at all, in CBA/Jmice, whereas they grew readily in athymic (nude) mice. Theincrease in immunogenicity of two out of three of these latterclones was accompanied by expression of the class I H-2Dkmajor histocompatibility complex antigen that was not detect-able in the parental SP1 cells. At least some of these resultswould appear to be due to exposure to Ca3(PO4)2 alone, as wefound that it resulted in 5/20 (25%) clones manifesting meta-static properties. Our results suggest that heritable changes inmalignant behavior of transfected tumor cells can be observedat high frequency subsequent to the process of Ca3(PO4)2-mediated DNA transfection, and these changes may be broughtabout in part by inherited disturbances in expression ofrecipient cell genes.

The advent of gene transfection techniques has had anenormous impact on developing an understanding of genestructure and function. This is particularly true in the field ofcarcinogenesis, where the functions of oncogenes or putativeoncogenes have been dissected in depth by the use of suchprocedures (1, 2). More recently, gene transfection tech-niques have also been employed to study aspects of tumorprogression and metastasis at the genomic or gene level (3).A potential problem with gene transfection studies stemsfrom the possibility that elements of the transfection andsubsequent selection procedures themselves may cause ac-quired phenotypic alterations. The magnitude of this problemmay not be so serious-or apparent-in tumorigenic trans-formation studies using immortalized cell lines, where one isconcerned with the emergence of tumors and in which,presumably, the transfer of an oncogene is necessary for

development of the neoplastic phenotype. But in situations inwhich alterations in existing malignant behavior are underscrutiny, the inherent genetic instability of tumor cells (4)may make them particularly vulnerable to genetic alterationsinduced by DNA transfection techniques and subsequentselection procedures.With regard to the latter possibility there are two especially

noteworthy points. First, it has already been shown thattransfection of plasmids containing oncogene sequences intonontumorigenic fibroblastic cells can result in secondarychromosomal changes that lead to neoplastic transformationwithout stable integration of the transfected oncogene (i.e.,"hit-and-run" tumorigenesis) (5). There are also examples of"insertional mutagenesis," usually involving stable insertionof viral or proviral sequences into the genome of the recipientor infected cells (e.g., see refs. 6-8). Second, although onemight assume that such events would be very rare at anygiven locus, it is now apparent that conventional mutagenicagents can sometimes cause very high frequency heritablephenotypic alterations due to inherited disturbances in geneexpression at single gene loci (9, 10). Among the possiblemechanisms that could bring this about is induced changes inDNA methylation (11-13). Thus, if the procedure of DNA-mediated transfection is directly mutagenic, then, like othermutagens, it may also cause, or be associated with, inheritedchanges in gene expression at frequencies far higher thanthose normally associated with classical mutations.

In this paper we present evidence that Ca3(P04)2-mediatedtransfection of a nonimmunogenic and very poorly metastaticaneuploid mouse mammary adenocarcinoma (the nature ofwhich is described in ref. 14) is associated with the emergenceof a high frequency of either less aggressive or more aggres-sive variants, and the changes are heritable. As such, theresults have important implications for the study of neoplasiaand control of gene expression in general, and tumor pro-gression/metastasis studies in particular, using gene trans-fection techniques.

MATERIALS AND METHODSTumors. CBA.SP1 (abbreviated SP1) is a nonmetastatic

infiltrating mammary intraductal adenocarcinoma that arosespontaneously in an 18-month-old female retired breeder ofthe inbred CBA/J mouse strain. The characteristics of thisaneuploid tumor (which has a mode of 68-72 chromosomes)have been described in detail elsewhere (14). Subcutaneousinoculation of 105 uncloned SP1 cells in the lower right-handflank leads to progressively growing encapsulated tumors.

Abbreviations: MHC, major histocompatibility complex; G418r,resistant to the antibiotic G418; tum-, nontumorigenic; FACS,fluorescence-activated cell sorter.tTo whom reprint requests should be addressed.

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Animals die of large "primary" tumors between 5 and 8weeks after inoculation. No macroscopic metastases haveyet been observed in CBA/J (or nude) mice given 10'uncloned SP1 cells subcutaneously.

Mice. CBA/J mice, purchased from The Jackson Labora-tories, were used as well as 8- to 12-week-old female athymicnude (nu/nu) mice on a BALB/c background purchased fromHarlan Sprague-Dawley (Indianapolis, IN). In general, 8- to12-week-old female CBA/J mice were used, though in someexperiments either 8- to 12-week-old male CBA/J mice or6-month-old retired female breeders were used.DNA Transfections. DNA transfection was performed ba-

sically according to the method of Graham and van der Eb(15) as modified by Wigler et al. (16). Briefly, 100-mm disheswere seeded with 6 x 105 cells 16-24 hr before transfection.Cells were either transfected with pSV2neo (17) or cotrans-fected with pSV2neo plus pT24-c3 (18), which carries thehuman T24 bladder carcinoma Ha-ras oncogene, at a molarratio of 1:10. No carrierDNA was used in these transfections,which incorporated a glycerol shock (19). Cells were subcul-tured into G418 (GIBCO; 400 ,g/ml) selective media 48 hrafter DNA transfection, and drug-resistant (G418f) colonieswere harvested 14-17 days later.DNA Extraction and Filter Hybridization. High molecular

weight genomic DNA was extracted from cultured cells asdescribed (20). Solid primary tumor tissue was pulverized inliquid nitrogen by using a mortar and pestle prior to DNAextraction. DNA digested with restriction endonuclease was

separated by electrophoresis, transferred to nitrocelluloseaccording to the method of Southern (21), prehybridized, andhybridized as previously described (20) to a random primedprobe (22).

Antisera and Immunofluorescence. The monoclonal anti-bodies used were as follows: anti-Kb/Db [clone 20-8-4s (23)],anti-Kk [clone 16-3-22 (24)], anti-Dk [clone 15-5-5s (24)], andanti-K/D [clone M1/42, which is specific for a nonpolymor-phic determinant on all class I (K/D) haplotypes (25)]. Detailsof these monoclonal antibodies have been published else-where (14). Immunofluorescence detection procedures werecarried out as previously described (14).

RESULTS

Transfected Cells Are Capable of Metastatic Growth. Table1 reports the results of two experiments in which SP1 cellswere either transfected with the pSV2neo plasmid alone or

cotransfected with pSV2neo plus pT24-c3, a plasmid carryingthe human T24 bladder carcinoma Ha-ras oncogene (18). Ourapproach was similar to that described by others in relation-ship to transfection of Ha-ras into 3T3 cells, primary embryofibroblasts, or mouse mammary tumor cells (3, 26, 27). In thefirst experiment 5-10 transfected G418' colonies were iso-lated, pooled, and grown to mass culture. Approximately 105of these cells were injected subcutaneously into syngeneicmice to assess their capacity for metastatic growth. As shownin Table 1, the pSV2neo + Ha-ras cotransfected cells gaverise to lung metastases in 85% of the animals tested. A highincidence of metastasis was also observed after inoculation ofsingle Ha-ras-transfected SP1 clones, all of which expressedtransfected ras sequences at various levels (unpublishedresults). A significant number of animals (17%) inoculatedwith cells transfected with pSV2neo alone also gave rise tometastases, whereas none were detected after subcutaneousinjection of the uncloned parental SP1 cell line. In the secondexperiment (Exp. 2 in Table 1), in which the inoculumcomprised a larger pool of individually derived pSV2neotransfectants (50-100 colonies), virtually every animal de-veloped metastases. If it is assumed that only a fraction ofSP1 cells acquire metastatic potential after transfection withpSV2neo, then it follows that the likelihood of detectingmalignant cells would increase when the inoculum compriseda larger number of individual transfectants. Since a G418r cellline (SP1 neoKi; Table 1) derived from a metastatic tumorwas found to be highly metastatic after inoculation intosyngeneic mice, we conclude that at least some of themalignant cells generated after transfection and drug selec-tion have stably acquired a metastatic phenotype. In all theseexperiments the number of tumor nodules in the lungs wasmodest-between 1 and 10-and the nodules were approx-imately 1-2 mm in diameter at 6-8 weeks after tumor cellinoculation (Table 1). This is not unusual for spontaneousmetastasis of epithelial tissue tumors (carcinomas).pSV2neo Metastases Result from a Limited Number of

Transfected Cells. Southern analysis of mass cultures ofpooled pSV2neo-transfected colonies and cultUres derivedfrom individual metastases supports the premise that only asmall number of pSV2neo-transfected clones are capable ofmetastatic growth. As shown in Fig. 1, all of the metastatictumors derived from pSV2neo-transfected cells retainedplasmid sequences. Digestion of genomic DNA with BamHI,a single cutter of pSV2neo, generates two unique fragmentsfor each insertion and a 5.8-kilobase (kb) fragment, reflecting

Table 1. Metastatic properties of SP1 mammary carcinoma pooled transfectants

No. of G418r No. of No. of mice Mice withcolonies mice with primary metastases

Exp. Transfected DNA selected injected tumor No. %o1 None 10 10 0 <1

pSV2neo (1 ug) 5-10 14 12 2* 17pSV2neo (1 ,ug) + pT24-c3 (10 /ig) 5-10 14 13 11 85

2 pSV2neo (10 ug) 50-100 4 4 4 100

SP1 neoK1 4 4 3 75

SP1 cells were transfected as indicated; the concentration of DNA added to each 100-mm dish (6 x 101 cells per dish)is given in parentheses. After DNA transfection and selection, G418r colonies were pooled and 10i cells were injectedsubcutaneously into syngeneic CBA/J mice; 101 cells from the uncloned SP1 parent line were injected as a control. Growthof primary tumors was monitored weekly, and after 6-8 weeks animals were sacrificed and examined for visible lung, liver,or kidney metastases. In Exp. 2 but not in Exp. 1 cells were shocked with glycerol 5 hr after addition of Ca3(PO4)2/DNAcoprecipitate.*Metastases were detected in the lungs of one mouse arnd in the kidneys of a second. In all cases in which lung metastaseswere detected in these experiments, and other experiments reported in this paper, between 1 and 10 discrete nodules,approximately 1-2 mm in diameter, were observed 6-8 weeks after inoculation.tSP1 neoK1 is an established cell line derived from the kidney metastasis obtained in Exp. 1 after inoculation ofpSV2neo-transfected cells. The results with SP1 neoK1 show that the metastatic phenotype is heritable in this G418rtransfectant.

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EXPT. 1

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FIG. 1. Southern hybridizationofgenomic DNA. A 5-,ug sample ofDNA was digested with BamHI(New England Biolabs), electro-phoretically fractionated throughgels of0.8% agarose in TBE (90mMTris borate, pH 8.5,5 0mM EDTA)and transferred to nitrocellulose fil-ters. Linearized pSV2neo was la-beled by random priming to a spe-cific activity of2-5 X 108 cpm/tg ofDNA for use as a probe. Filterswere hybridized as described (20)and washed twice at room temper-ature with 2 x SSC + 0.1% sodiumdodecyl sulfate (NaDodSO4) (1xSSC = 150 mM NaCl/15 mM sodi-um citrate) followed by two cyclesof alternating washes in 2x SSC/0.1% NaDodSO4 and 0.2x SSC/0.1% NaDodSO4 at 50°C. Filterswere then air dried and exposed toKodak XAR-5 film with CronexLightning Plus intensifying screensat -70°C for 16-24 hr. Analyses ofcell inocula, primary (10) tumors,and metastases from pooled trans-fectants (A) and a single isolatedclone SP1 neo5 (B) are shown.

tandem copies. Analysis of the cell inoculum from Exp. 1(Table 1, Fig. 1) revealed three strongly hybridizing bandsand a few weaker bands, suggesting that at the time ofinjection the pooled population contained only a single or asmall number of independent transfectants. The two meta-static tumors isolated in this experiment show the samehybridization pattern as the inoculum, whereas a primarytumor taken from an animal that did not develop metastasescontained two additional strong hybridization bands thatwere not detected in the initial inoculum. These bands mayrepresent a minor subset of the original population, orperhaps they arose by DNA rearrangement. As expected, theinoculum in Exp. 2 (Table 1, Fig. 1), which comprised 50-100independent transfectants, manifested a much more compli-cated pattern of hybridizing bands. Metastases from threeinoculated mice showed the same two hybridizing bands,suggesting that in each case the same cell grew in the lungsof these animals. The selection of this cell was apparent evenwithin the primary tumors from these mice. Even more

remarkable is the finding that these metastases have theidentical two hybridizing bands as one of the independent

clones isolated from the same transfected cell population(Fig. 1B). This clone, SP1 neo5, is 1 of the 3 clones found tobe metastatic out of 16 tested (see below). These data arguestrongly for a selective growth advantage both in vitro and invivo for malignant cells, as shown, or implied, in a number ofother systems (28, 29).

Transfected Clones Have Altered Metastatic or TumorigenicProperties at a High Frequency. To determine the frequencyof metastatic clones within the population of transfectedcells, individual colonies were isolated after selection in G418following transfection with pSV2neo (Table 1, Exp. 2).Newly cloned cultures of SP1 were used as a control toexclude the possibility that metastatic variants exist at a highfrequency within the parent population. Sixteen independentG418r clones arising from transfection with pSV2neo and 20individual SP1 clones were assessed. The results, given inTable 2 and summarized in Table 3, confirm and extend ourresults obtained with the pooled cultures. Thus, 3 of the 16pSV2neo-transfected clones were able to metastasize to thelungs, whereas none of the 20 SP1 clones showed any sign ofmetastatic growth.

Table 2. Metastatic and tumorigenic properties of selected untransfected and pSV2neo-transfected clones in syngeneic

and nude mice

No. of mice with primary tumor/ No. of mice with lung metastases/no. of mice injected no. of mice with primary tumor

CBA/J nu/nu CBA/J nu/nu Phenotype*

Clone Exp. 1 Exp. 2 Exp. 2 Exp. 1 Exp. 2 Exp. 2 of clone

SP1 22 0/3 5/5 4/4 0/5 0/4 tum+ met-SP1 37 1/3 5/5 4/4 0/1 0/5 0/4 tum+ met-SP1 neoS 5/5 5/5 5/5 3/5 2/5 2/5 tum+ met'SP1 neo22 3/3 NT 1/3 NT tum' met'SP1 neo23 3/3 5/5 NT 3/3 4/5 NT tum+ met'SP1 neo4 2/5 2/5 5/5 0/2 0/2 0/5 tum- met-SP1 neo24 0/3 0/4 4/4 0/4 tum- met-SP1 neo25 0/3 0/4 3/4 0/3 tum- met-

Individual SP1 and SP1 pSV2neo-transfected clones were assessed for growth in syngeneic CBA/J and nude mice as inTable 1. Exps. 1 and 2 were independent. Results for clones that displayed altered metastatic or tumorigenic properties aregiven here. NT, not tested.*Phenotypes: tum+, tumorigenic; tum-, nontumorigenic; met+, metastasizing; met-, nonmetastasizing.

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Table 3. Summary of metastatic and tumorigenic properties ofvarious sources of cloned SP1 cells

No. of No. oftum- clones/ met' clones/

Source of clones no. of no. oftested clones tested clones tested

SP1 clones (nontransfected control) 0/20 0/20SPi clones obtained after Ca3(PO4)2

treatment 0/20 5/20SP1 pSV2neo-transfected clones 3/16 3/16SP1 pSV2gpt-transfected clones 1/18 3/18SP1 clone 31 pSV2neo-transfected

clones* 1/6 0/6SP1 clone 32 pSV2neo-transfected

clones* 0/6 2/6SP1 clone 33 pSV2neo-transfected

clones* (l)t/6 1/6

*Three individual clones of SP1 were transfected with pSV2neo andsix G4181 colonies from each were grown to mass culture andassessed for their tumorigenic and metastatic growth in vivo.

tThis line is still unconfirmed as an authentic tum- clone.

In addition to the high frequency of metastasis-positivetransfected clones observed, our analysis unexpectedly re-vealed that 3 of the 16 pSV2neo G418r clones were consid-erably less (or non-) tumorigenic in syngeneic CBA/J mice-i.e., they behaved as tum- clones (14). We also noted that 3of the 20 SP1 clones obtained by simple in vitro cloning alsogrew poorly when first tested in groups of three CBA/J mice.These clones were not "authentic" tum- clones, however,because the phenotype was not reproducible when cells fromthe same frozen batch of cells were thawed, grown to massculture, and then retested. This type of variation has beenseen for a variety of parameters and is probably due torandom and unstable clonal variation (30). In contrast, the 3tum- pSV2neo clones maintained their tum- phenotypewhen similarly retested. However, these tum- pSV2neoclones grow normally in 100% of nude mice, suggesting animmunological basis for reduction of their tumorigenicity inCBA/J mice.

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The observed effects on the metastatic and tumorigenicphenotype of SP1 cells are not specific to transfection withpSV2neo, as similar results were obtained after transfectionand selection with pSV2gpt (Table 3). Indeed, treatment ofSP1 cells with Ca3(PO4)2 for 16 hr prior to cloning yielded 5metastatic clones out of a total of 20 (i.e., 25%). However, noauthentic tum- clones have yet been observed after Ca3-(PO4)2 treatment alone. Although the number of individualclones tested was limited, we have also found that bothnontumorigenic and metastatic clones were obtained aftertransfection of independent SP1 clones with pSV2neo (Table3). This argues against the possibility that the process ofDNAtransfection and selection inadvertently selects for raremetastatic or nontumorigenic variants within the unclonedparental SP1 population.

tum- Clones Are Often Associated with Increased Expres-sion ofMHC Class I Antigens. The development of tum- cellsafter DNA transfection is reminiscent of our previous find-ings with agents such as ethyl methanesulfonate and 5-azacytidine (14, 31, 32). The tum- phenotype in these latterstudies was often found to be associated with an increase inclass I MHC (H-2) surface antigens (14). On analysis of thetum- clones generated after DNA transfection and selectionof pSV2neo colonies, we found that once again the tum+tum- phenotypic change was associated with an alteredexpression ofMHC class I antigens. Samples of SP1 and SP1neo+ clones were screened by fluorescence-activated cellsorter (FACS) analysis for MHC expression in a double blindexperiment. All but two clones were found to be H-2negative, like the SP1 parental cell line. The two H-2-positiveclones were identified as two of the three tum- pSV2neoclones-i.e., SP1 neo4 and SP1 neo24. Fig. 2 shows arepresentative sample of the FACS analysis of SP1 cells andthree clones, two of them being authentic tum- clones (SP1neo4 or neo24). As in previous studies (14) using alkylatingagents or 5-azacytidine, it was the H-2Dk antigen, not theH-2Kk antigen, that was expressed.

DISCUSSIONOur results clearly indicate that the procedure of Ca3(PO4)2-mediated DNA transfection of tumorigenic cells can result in

SP1-Neo-24

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RELATIVE FLUORESCENCE INTENSITY (ARBITRARY UNITS)

FIG. 2. Immunofluorescence analysis of class I MHC (H-2) antigen expression of SP1 cells after Ca3(P04)2-mediated transfection ofpSV2neoand selection for G4181 colonies that manifested a tum- phenotype. Eight different clones were analyzed by FACS, as well as the uncloned SP1nontransfected parent. Irrelevant anti-Kb/Db antibody (aKb/Db) served as a negative control. The results obtained on SP1 and three of the clonesare shown above. SP1 refers to the uncloned SP1 (nontransfected parent); SP1-22 is a recently derived clone of SP1 (nontransfected) that initiallydid not grow in three CBA/J mice but did so upon retesting of frozen and thawed cells; SP1 neo4 and SP1 neo24 are cell lines established fromindependent G418T colonies (clones) obtained after transfection with pSV2neo. All other SP1 clones were found to be H-2Kk- andH-2Dk-negative, as were G418r SP1 transfectants that manifested a tumorigenic (tum+) phenotype in CBA/J mice. The H-2Dk-positive phenotypewas found in 2/3 pSV2neo SP1 transfectants-i.e., the ones shown above.

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heritable alterations in both the tumorigenic and malignantbehavior of the transfectants at a high frequency. Using thehighly tumorigenic but nonmetastatic mouse mammary adeno-carcinoma, SP1, we have observed that transfection of SP1cells with pSV2neo alone gave rise to lung metastases in asignificant number of animals. Analysis of independentpSV2neo-transfected G4181 clonal isolates revealed thatalmost 20% of these clones manifested metastatic potential.We also noted that 3/16 of pSV2neo transfectant clonesshowed an increased immunogenic phenotype in that theyhad a greatly impaired ability to grow in syngeneic CBA micewhile being able to grow in 100% of athymic (nu/nu) mice.The latter observation was strikingly reminiscent of ob-

servations indicating that treatment of tumor cell lines withhighly mutagenic alkylating agents, such as ethyl methane-sulfonate or poorly mutagenic agents that can alter geneexpression, such as 5-azacytidine, can result in very highfrequencies (between 10% and 90%) of tum- clones (31, 33).Indeed, this has been observed with the SP1 mammarycarcinoma itself (14, 32), in which it was also found that tum-SP1 clones manifested an increase in the expression of selfclass I MHC antigens, specifically of H-2Dk (14). As shownin this study, 2/3 pSV2neo clones also similarly displayedelevated levels of the H-2Dk antigen.The changes in H-2 antigen expression are important with

respect to the possible mechanisms involved in the phenom-enon. Because of the random nature of the insertion oftransfected DNA, the high frequency of changes observed atspecific gene loci (e.g., H-2D) cannot be accounted for byspecific insertional events. On the contrary, it is indicative ofwidespread alterations in gene regulation such as those thatoccur through DNA methylation, gene amplification, orrearrangements. Whatever the underlying mechanism(s), itremains to be determined to what extent changes in cellularphenotype brought about by DNA transfection are influencedby such factors as the nature of the recipient cell line and thetransfection protocol used. With regard to the former, thesusceptibility to the types of phenotypic alterations describedhere may clearly be a function of the extent of geneticinstability in a particular cell line that is used as a recipient forgene transfection. Thus the nature and extent of the changeswe have documented with SP1 mammary carcinoma cellsmay not necessarily be observed when other cell lines areused as DNA recipients (e.g., see ref. 3).With respect to which components of the transfection

procedure are important in effecting these phenotypechanges, our results indicate that treatment of SP1 cells withCa3(PO4)2 alone can lead to alterations in the metastaticpotential of these cells, indicating this procedure can accountfor at least some of our results. The absence of detectabletum- clones after Ca3(PO4)2 treatment alone may mean thatit is irrelevant to the induction of this phenotype or thatCa3(PO4)2 treatment in combination with some other com-ponent of the DNA transfection process (e.g., G418 selec-tion) may be necessary to generate tum- clones at highfrequency. It should be noted that Rubin and co-workershave described results showing both growth-stimulating (pro-liferative) effects and morphological changes on BALB/c3T3 cells by precipitates of calcium ortho- or pyrophosphate(34-36).Our results have broad implications for the interpretation

of experimental results involving gene transfection proce-dures, since it is obvious that in some cases the observedphenotype of transfectants may not necessarily be a mani-festation of the product of the transferred gene. In respect to

neoplasia it is noteworthy in our experiments that we choseto (i) examine metastasis rather than just tumorigenicity; (ii)use normal immunocompetent mice as well as, or instead of,nude mice; and (iii) test clones as well as pooled colonies ofG418r cells. As a result, we detected gross changes in themalignant, tumorigenic, and immunogenic status in many ofthe transfectants, some ofwhich would have gone undetectedhad we confined our analysis to the relative tumorigenicity ofpooled colonies in nude mice.

We thank Astrid Eberhart for her excellent secretarial assistance,Micki Thomas, Dawn Coulson, and Bill Longhurst for their excellenttechnical help, and Dr. Louis Siminovitch for review of the manu-script. This work was supported by grants from the National CancerInstitute of Canada and the Medical Research Council of Canada (toR.S.K., M.L.B., and B.E.) and the U.S. National Institutes ofHealth (lRO1-CA41233). R.S.K. and B.E. are Research Associatesof the National Cancer Institute of Canada, and M.L.B. is a ResearchScholar of the Medical Research Council.

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