[CANCERRESEARCH54,3889-3896,July15,1994J

ABSTRACT

Wehaveexaminedthecontributionofthe mitochond.rialgenometo thetumorigenic phenotype expressed by human cell lines derived from anovarian and a cervical carcinoma and from an osteogenic sarcoma. Allthese continuous cell lines are anchorage-Independent in soft agar andform tumors in athymic nude mice. Long-term exposure of the cells toethidium bromide, an Intercalating agent which Inhibits mitochondrialDNA replication,gave rise to subclonesdepletedof mitochondrialDNAand RNA molecules and displaying either anchorage independence ordependence. These respiratory-deficient subclones contain disorganizedand enlarged mitochondria, are auxotrophic for uridine and pyruvate,and grow in vitro at a rate nearly identical or moderately slower than theirrespective parent. The tumor-forming ability of both anchorage-independent and -dependent cell lines was tested by s.c. and intramuscularImplantation of the cells in nude mice. We found that the tumorigenic

capacity was influenced by the route of inoculation. Subcutaneously,mitochondrial DNA-less cell lines are either poorly or nontumotigenic,while all but one cell line form tumors when implanted into the hind legmusde The relative in vivo growth rate of the parent and the mitochondrial DNA-less subclones reflects their respective in vitro rate of growth.All intramuscular tumors introduced into culture mimic the molecularand phenotypictraits of the Injectedcells, with the exceptionof theanchorage-dependent cell lines which give rise to anchorage-independenttumor cell lines. The present observations indicate that human cellswithout mitochondrial DNA have the capacity to proliferate and formtumors in vivo.

INTRODUCTION

The mitochondrial genome (mtDNA)4 of vertebrates contains asimilar set of genes involved in oxidative phosphorylation and dccIron transport (reviewed in Ref. 1). Point mutations or gross alter

ations in mtDNA have been shown recently to accompany manyhuman pathologies (2—4).Whether such mutations could also contribute to the tumorigenic phenotype of human and other vertebratecells has not yet been established, but alterations in the mtDNA ofhuman oncocytomas (5) and chemically induced rat hepatomas (6)have been reported. In addition, a large body of evidence obtainedthrough somatic cell genetic studies (reviewed in Refs. 7 and 8),

chemically induced tumors (9, 10), and examination of the cell surfacecharacteristics of petite yeast mutants (11) suggest the involvement ofmtDNA in the cancer phenotype. Models of carcinogenesis based onmtDNA mutations have been proposed (12, 13).

Recently, we have examined the contribution of the mitochondrialgenome to the tumorigenic capacity of transformed avian cells.

Received 12117/93;accepted 5/11/94.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.

1 This work was supported by a grant to R. M. from the Cancer Research Society, Inc.,Montréal.H. W. is the recipient of a fellowship from the Cancer Research Society, Inc.

2To whom requests for reprints should be addressed, Départementde biochimie,Facultéde M6decine, Universitéde Montréal,C.P. 6128, Succ. A, Montr6al, QuébecH3C 3J7, Canada.

3 Present address: Ceiltech, Ltd., 216 Bath Road Slough, SU 4EN, Berkshire, United

Kingdom.4 The abbreviations used are: mtDNA, mitochondrial DNA; mtDNA-less, devoid

of mtDNA molecules; agar', anchorage-independent; agar, anchorage-dependent;ATCC, American Type Culture Collection; GAPDH, glyceraldehyde-3-phosphatedehydrogenase.

mtDNA-less cells, i.e., cells devoid of mtDNA molecules, were firstdeveloped following long-term treatment of primary chick embryocells (14) and tumorigenic avian cells (15, 16) with ethidium bromide,a well-known inhibitor of mtDNA replication and transcription (17).In cultured cells, ethidium bromide intercalates into mitochondrialDNA and RNA and nucleolar ribosomal RNA but insignificantlywithin the chromatin network (18). These cells were used to investigate their capacity to grow detached in semisolid agar media and toinduce tumors at the site of injection in the wing web of day-oldchicks. Anchorage-independent avian cells rendered mtDNA-less losethe ability to form colonies in soft agar and to proliferate in day-oldchicks (19, 20). In contrast, cytoplasmic hybrids resulting fromcrosses between the mtDNA-less cells and cytoplasts from enucleatedparental cells recover mtDNA along with the capacity to grow in softagar and induce tumors at the site of injection. Cybrids constructedwith cytoplasts from enucleated nontumorigenic chick embryo cellsalso display similar tumorigenic properties.5 Overall, these observations were interpreted to indicate that mtDNA modulates the tumorigenic abilities of continuous transformed avian cells both in vitro andin vivo.

In the present article, we report on the tumorigenic capacity ofmtDNA-less human cell lines developed according to the experimental protocol established for avian cells. The parental human cell linesused were initially derived from ovarian (21) and cervical (22) car

cinomas and from an osteogenic sarcoma (23). We found that humancells depleted of mtDNA molecules could proliferate and form tumorsin athymic nude mice.

MATERIALS AND METHODS

Cells and Media. Three continuous human cell lines were used in thesestudies. The HeLa cell line was originally obtained from ATCC (clone CCL2)and kindly provided to us by Dr. V. Bibor-Hardy, Department of Medicine,Universitéde Montréal,Montréal,Quebec, Canada. HSL2, a subclone of theHeLa cell line, was isolated from semisolid agar medium with a Pasteur pipet.The 143N2 cell line is a subclone of the thymidine kinase-deficient osteosarcoma cell line 143B obtained from ATCC (clone CRL 8303). The 143N2 cellswere rendered resistant to geneticin following transfection with pSV2Neo. The2008 cell line was established from a serous carcinoma of the ovary. All celllines are anchorage-independent in soft agar and tumorigenic in male andfemale athymic nude mice.

Human mtDNA-less cells were developed essentially as described previously for primary and continuous avian cell lines (14—16).Briefly, HLS2 cellswere cultured in the presence of 50 ng/ml ethidium bromide for 90 days (24),and the 143N2 and 2008 cells were cultured in the presence of 180 ng/mlethidium bromide for 30 days. Following removal of the drug, cells wereseeded at low density onto plastic and individual colonies, ring-cloned, propagated, and screened for uridine and pyruvate auxotrophy. The 143N2 and2008 mtDNA-less subclones were developed by one of us (K. Z-P.) in thelaboratory of Dr. P. Andrews, Department of Pharmacology, GeorgetownUniversity, Washington, DC.

All cell lines were maintained in uridine (4.0 g.i@g/ml)-and pyruvate (110@g/ml)-containing Dulbecco's modified Eagle's medium (HSL2 and 143N2

cells) and RPM! 1640 (2008 cells) media supplemented with 10% fetal bovineserum. Penicillin (100 International units/mI), streptomycin (100 p@g/ml), and

5 R. Morals, K. Zinkewich-Péotti, and M. Parent, unpublished observation.

3889

Tumor-forming Ability in Athymic Nude Mice of Human Cell Lines Devoid ofMitochondrial DNA'

RéjeanMorals,2 Karen Zinkewich-Péotti,3 Manon Parent, Hong Wang, Feridoun Babai, and Max Zollinger

Départementsde biochimie[R. M., K Z-P., M. P., H. W., M. 1/ et de pathologie (F. B.], Facultéde Médecine@Universitéde Montréal,QuébecH3C 117, Canada

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Table 1 Descriptionofthe humancelllinesCell

linesParentmtDNA statusaCell

growthrequirements―

Uridine PyruvateSelective

mediumcChromosomecontent mean

(range)―Growthin soft

agareBrdUrdG-418HAT143N2

N2BhN2Zh143B

143N2143N2+

—

—N'

NY YY YR RR

RRS

SS101

(77—122)65(59—71)66(56—72)39.8

013.4N2T2'

N2BT1'N2BT2'N2ZT2'2008E3h.J

sri'E3T1aE3T2'143N2

N2BN2BN2Z

20082008E3B+

-

—

—

+—

+-

-N

NY YY YY YN NY YN NY YY YR

RRRR

RRRS

SSS102(94—113)

66(40-98)65 (48-73)72(65—112)59(52—65)58 (54-61)64 (55-87)60(57-66)59(53-64)20.7

23.534.947.137.5

030.830.8

22.8HSL2

HSAhHSEhL2T1'HeLa

HSL2HSL2HSL2+

—

—

+N

NY YY YN N61

(47—74)56 (50-65)58 (50—73)70(64—76)13.6

10.90

19.7

TUMORIGENICITYOF MITOCHONDRIALDNA-LESSCELLS

Fungizone (0.5 @g/ml)were routinely added to the culture media. Dialyzedserum (19) was used to prepare pyruvate- and uridine-depleted media. Cultures

were maintained at 37°Cin a humidified incubator with an atmosphere of 95%air:5% CO2. Cells were passaged twice a week, and the medium was changedevery other day. All cell lines were shown to be Mycoplasma free by theBoehringer Mannheim BM-Cyclin test.

In Vitro Growth Characteristics. The growth rates of the parental andmtDNA-less cell lines in media supplemented or not with uridine and pyruvatewere determined as reported previously for primary and continuous avian cells(16, 25). Cells were inoculated onto 60-mm plastic dishes, trypsinized atintervals, and counted (Coulter Counter). The cell medium was changed everyother day.

Cloning efficiencies in soft agar were determined as described (18) byculturing 1 X iO@cells harvested by trypsinization in mid-log phase per60-mm dish in growth media containing 0.33% Bacto-agar, 10% fetal bovineserum, pyruvate (110 @g/ml),and uridine (4.0 @g/ml).Cells were fed everyfive days with 2.0 ml of culture medium. At day 15, the number of colonieslarger than 0.1 mm in diameter was determined using an ocular micrometer onan inverted microscope.

Tumorigenicity in Nude Mice. The tumorigenicity of the parental and

mtDNA-less cells was assayed in 15- to 17-day-old and 6-week-old malecongenitally athymic nude mice. Cells in mid-log phase were trypsinized,suspended in growth medium at 7 X 10@-1X iO@cells/mI, unless otherwiseindicated, and injected (0.1 ml) either s.c. in the flank or intramuscularly intothe right hind leg. After injection, the cells remaining in the syringe needlewere always >85% viable as determined by 0.2% trypan blue dye exclusion.

Mice were kept for more than 6 months after injection before scoring asnontumorigenic. With some exceptions, the parental and mtDNA-less cell lines

produced palpable tumors within the first 8 weeks at the site of inoculation.Mice were killed by ether anesthesia followed by cervical dislocation, and theexcised tumors were placed on filter paper to measure their length, width, andthickness. When characterization of the tumor cells was desired, tumors were

excised under sterile conditions, minced with scalpels, and incubated in Dis

pase for 30 ruin before plating in growth medium. Tumor cells derived fromcell lines 143N2 (N2T2), N2B (N2BT1 and N2BT2), and N2Z (N2ZT2) weretested for drug resistance to 5-bromodeoxyuridine and geneticistand sensitivityto hypoxanthine/aminopteriWthymidine medium. Cells derived from tumorsinduced by cell lines 2008 (8T1), E3 (E3T1 and E3T2), and HSL2 (L2T1) werecultivated for over a month before being tested for their capacity to grow in

uridine- and pyruvate-free medium.

Distribution of Chromosome Number, Histological Evaluation, and

Electron Microscopy. For chromosome analysis, cells of subconfluent cul

tures were arrested in metaphase with 0.2 pg/mi Colcemid, allowed to swell in75 mM KC1, and fixed in cold methanol:acetic acid (3:1). Air-dried metaphase

spreads were stained with 4% Giemsa stain and photographed under a lightmicroscope. Unless otherwise indicated, the number of chromosomes in atleast 10 good metaphase spreads was determined for each cell line.

For electron microscopy, subconfluent monolayers were harvested by scraping and processed as described (16).

For light microscopy, tissue samples taken from different parts of theexcised tumors were fixed in 4% neutral buffered formalin and embeddedin paraffin. Sections cut at 5 @.tmwere stained with hematoxylin-phloxinesaffron.

Extraction of DNA from Mitochondria. HSL2 cells in late log phasewere trypsinized, resuspended in cold deionized water, and homogenized.After addition of sucrose to 0.29 M, a crude mitochondrial fraction wasobtained by differential centrifugation. The organelles were lysed by the

addition of sodium dodecyl sulfate, and mtDNA was purified by CsClethidium bromide gradient centrifugation as reported previously (14). ThemtDNA concentration was estimated by electrophoresis on agarose gels.

Extraction of Total Cellular RNA and DNA. Cells (5 X iO@to 1 X 108)harvested by trypsinization were used to prepare DNA and RNA Total cellularRNA was isolated by a variant of the guanidium isothiocyanate-CsCl methodwhich includes Sarkosyl in the homogenizing buffer (26). The integrity of theRNA was assessed by ethidium bromide staining. Total cellular DNA wasobtained as described (15). The concentration of RNA and DNA solutions inwater was determined by absorbance.

Cloning of Human Mitochondrial DNA. HeLa cell mtDNA was doubledigested with Hint/Ill and XbaI and ligated into phagemid pBluescript (SK).The recombinant molecules were used to transform Escherichia coli strainXL1. Clones were selected by growing the ampidilin-resistant colonies ontetracycline + ampicillin plates, and one of the colonies was chosen to givepMtH3. The recombinant phagemid was isolated by alkaline lysis, and thenucleotide sequence of the cloned mtDNA fragment was determined by thedideoxynucleotide method of Sanger et aL (27). The fragment is 1423 nudeotides long and spans the region between nucleotides 10257 and 11680 of thereported human mtDNA sequence (28). Parts of the ND3 and ND4 genes andthe entire tRNA@ and ND4L genes are contained within this region; the NDgenes encode protein elements found in complex I of the mitochondrialenergy-generating pathway.

a@ contains mtDNA; —,devoid of mtDNA.b Uridine, 4.0 @g/m1; pyruvate, 1 10 mg/mI.

CBromodeoxyuridine(BrdUrd), 100 @g/m1;geneticin(0-418), 800 @g/ml;100gtathypoxanthine-5.5g.&siamethopterin-13.5@.tMthymidine.d At least 10 metaphase spreads were counted for each cell line, except for N2T2 cells where the number is 5.

C Percentage of cloning efficiency. Values are the mean of at least two experiments done in duplicate.

1N, not required; Y, required.gR,resistant;5, sensitive.1.Colonies were ring-cloned following long-term treatment of 143N2, 2008, and HSL2 cells with ethidium bromide.ICellpopulationsderivedfromdifferentexcisedtumorsinducedin nudemicebytheparentalcellslisted.J Equivalent to subclone 2008.Et3 (K. Zinkewich-Péoui and P. Andrews, manuscript in preparation).

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TUMORIGENICITY OF MITOCHONDRIAL DNA-LESS CELLS

Large scale preparation of phagemid DNA was performed as reportedpreviously (16). Plasmid DNA was cleaved with HindIll and XbaI, anddigested materials were fractionated by size on a 1.0% agarose gel. The bandcontaining the mtDNA fragment was excised from the gel, and the materialwas adsorbed on a silica matrix provided with the Geneclean Kit and eluted asrecommended by the supplier. The DNA concentration was estimated by

electrophoresis on agarose gels.Southern and Northern Blot Hybridizations. Restricted total cellular

DNA samples were fractionated by size on 0.7% agarose gels and transferredunder alkaline conditions onto nitrocellulose membranes as described (14).Total cellular RNA samples were fractionated by size on 1.0% agarose gels

under denaturing conditions (29) and transferred onto nylon membranes. Theblots were probed with random primed 32P-labeledhuman mtDNA and thepMtH3 mtDNA insert. Prehybridization, hybridization, and washing conditions were as recommended by the manufacturer of the ifiters. The filters wereblotted dry and autoradiographed at —70°Cfor various periods of time usingan intensifying screen.

Animals and Materials. Nude mice (nu/nu; CD-i) were obtained from

Charles River Laboratories (St-Constant, Québec,Canada). Growth media,

serum, penicillin, streptomycin, Fungizone, trypsin, and Colcemid were pur

chased from GIBCO Laboratories (Burlington, Ontario, Canada). E. coli strainXL! andphagemidpBluescript(SK) wereobtainedfromStratagene(LaJolla, CA). Restriction enzymes were purchased from Pharmacia Biotech, Inc.

(Montréal, Qu6bec, Canada) and used as recommended by the supplier.

[a-32PJdCFP was from Amersham (Oakville, Ontario, Canada), the Geneclean

Kit was from Bio 101 (La Jolla, CA), nitrocellulose filters were from

Schleicher and Schuell (Keene, NH), and Bacto-agar was from Difco Labo

ratories (Detroit, MI). All other chemicals were purchased from variouscommercial sources and were of the highest purity available.

A

.@ 165

.@ 12S

..* ND4L-ND4

am ND3

..@ GAPDH

Fig. 2. Northern hybridization of total cellular RNA from parental and derivedmtDNA-less cells. A, 20 @gof RNA extracted from parental (2008 and 143N2) andmtDNA-less (E3, E3T2, N2B, N2BT1, N2BT2, N2Z, and N2ZT2) cells were fractionatedby size on a 1% agarose-formaldehyde gel and blottedontoa nylonmembrane.The resultingfilter was@ with HeLa cell mtDNA. B, same filter as (A) but probed whh the mtDNA in

_7 sat Fepared from phagemidpMtH3.@ same ifiter as (A) but probed with a 925-basepairfragmeotofchkken@yceraIdehyde-3-phosphatedehydrogenase(GAPDH)comp1emenffiiy DNA.Exposure time: A and B, 20 h@ 7 days@Arm@i4iead@positions of the 16S and 125 rlbosomalRNAsandthe ND4L@ND4,ND3,and GAPDHmRNASieft size markei@Description of the celllinesisgiveninTable 1.

HindUI XbaI ScalA1@ @- II

,@, A@@A@'q,@(b@ Q@ Q@ ‘b@@ @b Q@@ 4@ Q@ ‘@

@u

@?3:F:ffU@*ij@pgJ1

7.5 —5.5

1.7 —

Fig. 1. Southern hybridization of total cellular DNA from parental and derivedmtDNA-less cells. Total cellular DNA (0.5 and 5.0 pg) from parental (143N2) andmIDNA-less (N2B, N2BT1, and N2BT2) cells, respectively, was restricted with Hind!!!,XbaI, and Sea!, fractionated by size on a 0.7% agarose gel, and blotted onto a nitrocellulosemembrane.A,DNASwereprobedwithHeLacellmtDNA.B,samefilteras(A)butprobed with the 1.4-kilobase mtDNA insert prepared from phagemid pMtH3. Exposuretime, 5 days. The number and size ofthe restriction fragments were as expected(34). Leftsize markers. Description of the cell lines is given in Table 1.

kb@ft/4@A.@ ‘47$@@,c@,/4@@9.5-

7.54.4-

2.4—

1.4W$,l1

1II.24

—9.5-

:

7.5-

4.4-

2.4-

1.4-

.24 —1

1—

,•I

1I

,II

RESULTS

Development and Characterization of mtDNA-less HumanCells. Following long-termexposureof HLS2, 143N2, and2008 cellsto ethidium bromide, the resulting populations were seeded at lowdensity in ethidium bromide-free medium. Well-delimited colonieswere ring-cloned, and those found auxotrophic for uridine and pyruvate were tested for their ability to grow in soft agar. The in vitro traitof anchorage independence has a strong correlation with tumor formation in vivo in mammalian systems (30, 31), although exceptionshave been noted (32—34).We found that ethidium bromide-treatedHSL2 and 143N2 cells gave rise to subclones that either grew (agar@)or not (agar) in soft agar, while all subclones derived from the 2008cell line were agar . Percentage cloning efficiency in soft agar of allagar@ subclones tested was found to be lower by 15 to 65% than thatof the parental cells. Extending the incubation period resulted in largercolonies but not in an increase in their total number. Agar subcloneswere unable to grow in soft agar, even when seeded at cell densities5 and 10 times higher. The agar@ and agar cell populations furthercharacterized in this study are listed in Table 1. Subclones N2B andN2Z were derived from cell line 143N2, subclones HSA and HSEwere from cell line HSL2, and subclone E3 was from cell line 2008.

The agar@ and agar subclones were found to be devoid of mtDNAmolecules. Analysis of mtDNA by Southern hybridization is illus

trated in Fig. 1 for cell line 143N2 and subclone N2B. Total mtDNA

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TUMORIGENICITYOF MITOCHONDRIAL DNA-LESS CELLS

(Fig. IA) and a cloned mtDNA fragment (Fig. 1B) were used to proberestricted fragments from total cellular DNA. In agreement withDrouin (35), human 143N2 mtDNA was found to be cleaved into theexpected number of fragments by HindIII, XbaI, and ScaI. Characteristic mtDNA bands were not seen in N2B cells on autoradiogramsof blots exposed long-term. The latter cells and mtDNA-less cell linesHSA, HSE, E3, and N2Z were also devoid of mtDNA transcriptshomologous to 16S and 12S rRNAs (Fig. 2A) and to the expected (36)mRNAs for ND4L-ND4 and ND3 (Fig. 28). All cell lines expressedGAPDH (Fig. 2C).

The mitochondrial ultrastructure of the mtDNA-less subclones resembled that of the previously described mtDNA-less avian cells (16).As shown in Fig. 3, a and d, mitochondria of parental 143N2 and 2008cells generally appeared elongated or oblong with transverse cristae,and the density of the mitochondrial matrix was greater than that ofthe surrounding cytoplasm. Marked structural changes were seen inmitochondria of agar@ (Fig. 3b) and agar (Fig. 3e) mtDNA-lesssubclones, i.e., reduction in the number ofcristae within the organelle,

loss of cristae orientation, and reduction of the electron opacity of themitochondrial matrix. In addition, we found that none of these celllines had detectable cytochrome c oxidase activity (data not shown),similar therefore to the situation in avian mtDNA-less cells (15, 16).Taken together, these observations indicate that long-term treatmentof human cells with ethidium bromide gave rise to cell populationsdevoid of a functional electron transport chain.

Fig. 4 shows the growth characteristics of parental and derivedmtDNA-less cell lines. The rate of growth of N2B and N2Z cells wasalmost the same as their parent (Fig. 44) and that of E3 (Fig. 4B) andHSA and HSE (Fig. 4C) was decreased by 20 and 60%, respectively.Upon removal of pyruvate and uridine, the proliferation of allmtDNA-less cell lines ceased after a few cell divisions, while thegrowth of the parental cells was unaffected. Auxotrophy for uridine

likely results from the inhibition of the flow of electrons along theelectron transport chain, preventing the oxidation of dihydrooroticacid to orotic acid (37). The requirement for pyruvate has not yet beendocumented but may be relatively nonspecific and reflect the need for

..,. ‘@, @; @; I E@1,@M .@ ‘ ;..,.. —@ -@@

Sr.,,@ @.@@,. . , ,@- . .,. •;.‘@ .@

@1@'@ a@@@ ,,@ j@@

@& @: •@-@@ ‘@!\@‘@

@@ @‘@k:@@―c

:2@:@

@:@ @*‘@

-@; ‘.@

,@ . ,

.@

-,r)

1%

. ‘4

Fig. 3. Appearance of mitochondria in sectionsof parental and derived mtDNA-less celis. Parentalcell lines: a, 143N2;@ 2008. MtDNA-less celllines: b, N2Z; c, N2ZT2; e, E3;)@E3T2. In mtDNAless cells, mitochondria appeared swollen with decreased amount of cristae, changes in orientationwithin the organelle, and mitochondrial profileswith circular cristae. Description of the cell lines isgiven in Table 1. X 20,000.

; @...@. ‘..@ ...@ “...@. .

.@@ •:@@ •@ •

@‘ .@ S..'

1.. I@ —I

@ 1pm

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100 200 300 400 500 100 200

Time (hours)

TUMORIGENICITYOF MITOdHONDRIALDNA-LESSCELLS

Tumors at the site of implantation were confirmed to be neoplasticby histological examination (Fig. 5). Both the parental and mtDNAless tumors were relatively well vascularized without evidence ofextensive necrosis or fibrosis. These observations suggest thatmtDNA-less human cells have the ability to induce capillary bloodvessel growth, which is an absolute requirement for a progressively

growing solid tumor (40). Moreover, these cells were also invasivesince intercellular and/or transcellular infiltration of the surroundingmuscle tissue were found (Fig. 5), suggesting the synthesis andsecretion by human mtDNA-less cells of one or more invasive factors.

The possibility that HSE cells may be tumorigenic but were rejected by the nude mouse has not been investigated further but appearsunlikely since no tumors developed in suckling nude mice which lacknatural killer cell activity. It may be that injected at other sites in nudemice, e.g., the cervix, the HSL2 and derived mtDNA-less cells coulddemonstrate a greater tumorigenic capacity than that shown here.

In Vitro Characterislics of Cultured Tumor Cells. Selected tumors induced by both agar4 and agar cell lines were introduced intoculture and used to analyze biological and biochemical parameters.No gross alteration of the chromosome content of the tumor cellsversus the parents was observed (Table 1). Furthermore, all tumor celllines tested were devoid of mitochondrial DNA and RNA molecules(Figs. 1 and 2), contained disorganized and swollen mitochondria(Fig. 3, c andf), and displayed either a similar (Fig. 4B) or moderatelyreduced growth rate (Fig. 44) as compared to the parents. All tumorcell lines derived from agar@ mtDNA-less cells showed a greatlyenhanced ability to form colonies in soft agar. More interestingly,agar mtDNA-less cells gave rise to tumor cells showing a highdegree of anchorage independence. Similar observations have beenreported with other mammalian systems (30, 41), suggesting that thein vivo passage of anchorage-dependent cell lines may select for rareanchorage-independent cells present in the population.

U)

0-—

14

0

14a)

Ez

DISCUSSION

This study was initiated to investigate a possible link between thetumor-forming ability of human cells and mitochondrial DNA. The

experimental procedure followed was developed previously to studythis problem in birds (19, 20) and involved the construction of celllines devoid of mtDNA molecules. This procedure has been used byothers recently to develop mtDNA-less human cell lines (42, 43). Inthe present study, we selected three continuous cell lines derived fromdifferent human tissues that were able to grow in nude mice and whichdisplayed an in vitro trait usually associated with tumorigenicity, i.e.,the capacity to grow in soft agar (anchorage independence). Longterm exposure of the cells to ethidium bromide gave rise to cellpopulations devoid of detectable mitochondrial DNA and RNA molecules. MtDNA-less human cell lines are auxotrophic for uridine andpyruvate and grow without a functional respiratory chain.

The development of mtDNA-less cells from human continuous cellpopulations indicates that the mitochondrial genome may not be

necessary to maintain immortalization, which is regarded as one of thepreliminary steps leading to cell tumorigenicity in vivo (44). Progression toward further growth autonomy is seen as anchorage-independent growth in most mammalian cell systems, although anchoragedependent tumongenic cell lines have been described (32, 45). Inbirds, we have shown that mtDNA modulates the anchorage-independent phenotype of continuous transformed cell lines (17). The progressive loss of mtDNA in the presence of ethidium bromide corre

lates with a decrease in anchorage-independent growth in soft agar.Long-term exposure to the drug led to the development of mtDNAless chicken cells which display anchorage-dependent growth, al

though rare anchorage-independent revertants could be selected from

3893

Fig. 4. Rates of growth of parental and derived mtDNA-less cells in the presence andabsence of uridine and pyruvate. Cells were grown in media containing 10% dialyzedserum. A, parental i43N2 cells (0); mtDNA-less cells N2Z (Lx), N2B (0), N2BT1 (A),andN2ZT2(Y).B,parental2008cells(U);mtDNA-lesscellsE3(0), andE3T2(A).C,parental HSL2 cells (El);mtDNA-less cells HSA(O)and HSE(@@).UrI@and Pyr@,mediasupplemented with 4.0 pg uridine and hO pg pyruvate/mI, respectively; Uri and Pyr,media devoid of uridine and pyruvate. Description of the cell lines is given in Table 1.

a substrate that can be used to oxidize NADH generated throughglycolysis. Indeed, we have observed that the glycolytic rate in avianmtDNA-less cells is 3 to 5 times higher than in the parental cells.5 Thepossibility that pyruvate serves as an alternate source of precursors forthe synthesis of various compounds in respiration-deficient cells hasalso been suggested (38, 39).

Tumorigenicity in Nude Mice. The ability of the parental andderived mtDNA-less cell lines to form tumors at the site of injectionin nude mice is shown in Table 2. The route of inoculation was foundto significantly influence the tumorigenic capacity of the human celllines. Injected s.c., 143N2 and 2008 cells formed tumors within4 weeks, while HSL2 cells, although anchorage independent, werenontumorigenic. When injected intramuscularly, all parental cell lines

formed tumors, but the latent period for HSL2 cells was generallygreater than for 143N2 and 2008 cells. The mtDNA-less cell lineswere either poorly or nontumorigenic when injected s.c. Except forHSE cells, intramuscular tumors were induced by all mtDNA-less celllines, although with delayed appearance as compared to the parents,

particularly for HSA cells. Tumor growth in nude mouse somewhatcorrelates with the in vitro rate of growth of the implanted cells.

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Table 2 Tumorigenicizyin nude miceof control and mtDNA-less humancellsTumorigenicityTake

incidence―mtDNArouteof injectionLatency

periodTumor

size(mm)―LengthWidthThicknessCell

linesParentstatus@im(weeks)cmean(range)143N2143B+2/2

6/62—47 (4—10)4 (2—6)3(1—5)N2B143N2—1,4e5/54—108 (5—10)5(3—8)4(2—5)N2Z143N2—0/35/57—98 (7—10)4 (3—6)3(2—5)2008+2/27/73—48 (4—10)3 (2—5)4(1-6)E32008—1/47/74—65 (2—10)5(3—7)2(1—3)HSL2HeLa+0/45/54—87 (4—10)5 (3—8)3(1—5)HSAHSL2—0/42/315, 184 (2, 6)2 (1, 3)1(1)HSEHSL2—0/40/4

TUMORIGENICITY OF MITOCHONDRIAL DNA-LESS CELLS

a@ contains mtDNA; —,devoid of mtDNA.b Number of mice with a tumor at the site of injection/number of mice injected. Mice scored as nontumorigenic were kept for more than 6 months after injection.CThe latencyperiodis the time requiredfor an intramusculartumor massto be detectedvisually or by palpation.No tumor regressionwaseverobserved.d Intramuscular tumors induced by 143N2 and 2008 cells were excised precisely 4 weeks after injection. Tumors induced by HSL2 and mtDNA-less cells were excised from 4 to

20 weeks after injection. The incidence of tumor formation in adult and suckling mice was the same.eThetumorderivedfromoneofthetwomiceinjectedwith1.5X iO@cells.

. ..:@@ ‘@. --@ .@ -.. . â€.̃@ .@‘.

.-@@@ @•-.-‘@.

- ,.. . . ,‘- . - ‘ -. -

@ ‘I-@@@

â€,̃@,.@ -

@-z@ @-@

. : .

@ ‘@@::-.

Fig. 5. Histological sections of intramusculartumors induced by parental and derived mtDNAless cells. Parental cell lines: a, 143N2; 4 2008.MtDNA-less cell lines: b, N2B; c, N2Z; e andf E3.All cell lines infiltrate skeletal muscles and showintercellular and transcellular invasion (arrows).Differentiation and morphology of parental andderived mtDNA-less tumor cells are similar. fa well-differentiated area in the center of an Btumor. Description of the cell lines is given inTable 1. X 800.

.@ j:.:@

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@ #@ ,,,s.e .a ;@. @• ,. @. .

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TUMORIGENICITY OF MITOCHONDRIAL DNA-LESS CELLS

the cell populations. In the present study, no attempt has been made tofollow as a function of time, the anchorage-independent capacity ofagar@ human cell populations treated with ethidium bromide. Anchorage assays of mtDNA-less human cell lines revealed cell populationsthat either grow or not in soft agar. We do not know if the human cellpopulations derive respectively from agar@ and agar cells aLreadypresent in the parental cell populations at the onset of the ethidiumbromide treatment, since not all parental cells may have the capacityto grow in soft agar, as suggested by cloning efficiency values (Table1). Very little is known concerning the genetic alterations required toinduce anchorage independence, although in some mammalian cellsystems a single gene seems to be sufficient (46—48).The number ofdifferent genes which when altered can make a cell anchorage

independent is not known. Genes encoding transforming growth factors, other mitogenic peptides, and specific cell surface componentsare among the likely potential candidates (49—51).Overall, the present observations indicate that the mitochondrial genome may not berequired to maintain the anchorage-independent phenotype of immortalized human cells.

The tumorigenicity of the human cell lines tested in this study isinfluenced by the route of implantation in nude mice. Such observa

tions have been already documented (52, 53), and tumorigenicity insusceptible animals is believed to be determined by both the intrinsic

properties of tumor cells and by host factors at the site of implantation(reviewed in Refs. 54 and 55). The subcutis and intramuscular microenvironment were found to support equally well the growth of celllines 143N2 and 2008. Both cell lines developed highly vascularizedtumors within 4 weeks at the site of inoculation. Injected into thesubcutis, their derived mtDNA-less cell lines, whether agar@ oragar, were poorly tumorigenic but grew relatively well when im

planted into the hind leg muscle. We found that the tumorigeniccapacity in subcutis of cybrids constructed between N2B and cytoplasts from enucleated osteosarcoma cells (HOS, ATCC and CRL1543) and normal human fibroblasts (WI38, ATCC and CCL 75) wasthe same as the parental cell line 143N2,5 suggesting a promotionalrole for mtDNA. mtDNA-less cells are auxotrophic for uridine andpyruvate, and the ability of these cells to grow intramuscularly could

be due, at least in part, to the higher level of vascularization of thistissue versus the subcutis. The circulating plasma uridine concentrations in the mouse and human range between 1 and 10 p.M (56),sufficient to promote in vitro, and likely also in vivo, the growth ofmtDNA-less cells. The circulating plasma pyruvate concentration in

mammals is the same as in most chemically defined culture media.The HSL2 cell line, a soft agar subclone of the parental HeLa cells,

was nontumorigenic when injected s.c., and this phenotypic trait wasconserved by subclones HSA and HSE, the derived mtDNA-less cell

lines. Implanted intramuscularly, HSL2 and HSA cells induced tumors at the site of inoculation but not HSE cells. These observationsare somewhat different than those reported recently by Hayashi et a!.(57)wheretheyshowedthatthetumorigeniccapacityof HeLacellsinoculated s.c. into the back of nude mice was lost by a derivedmtDNA-less subclone; other routes of inoculation were not tested.Introduction of mtDNA from normal human fibroblasts into mtDNAless cells restored s.c. tumorigenicity (57), similar therefore to thesituation in the chicken (20). In the present study, mtDNA-less celllines developed from HeLa cells appear to be less tumorigenic thansimilar cell lines from the 143N2 and 2008 cells. Other routes ofimplantation in nude mice should be used to test further the tumorigenic capacity of HSL2 cells and subclones HSA and HSE.

In conclusion, the present observations indicate that mtDNA-lesshuman cells derived from tumorigenic cell lines treated with ethidiumbromide can induce tumors in nude mice when inoculated at theproper site. These observations suggest that mtDNA may not be

necessary to maintain in vivo the tumorigenic phenotype of human

transformed cells. The possibility remains, however, that ethidiumbromide, a drug that shows mutagenic activity in bacteria and inducesunscheduled DNA synthesis in primary rat hepatocyte cultures (58),alters chromosomal DNA during long-term exposure of culturedhuman cells to the intercalating agent. Thus, it may be that themtDNA-less human cell lines developed in this study represent adaptative populations with characteristic proliferative behavior both invitro (see above) and in vivo. Mitochondria are being used as apotential target for anticancer drugs through interaction with mitochondrial macromolecular synthesizing systems (59, 60) and the respiratory electron transport chain (61, 62) or with the pyrimidine de

novo dihydroorotic acid dehydrogenase enzyme system (63). Whiledisruption of energy balance and respiration and inhibition of uridineprecursors represent antiproliferative mechanisms, the present observations suggest that such unique therapeutic avenues could not besufficient to totally eliminate human tumor cells in vivo.

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1994;54:3889-3896. Cancer Res   Réjean Morais, Karen Zinkewich-Péotti, Manon Parent, et al.   Lines Devoid of Mitochondrial DNATumor-forming Ability in Athymic Nude Mice of Human Cell

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