Clonal Diversity of the Kirsten-ras Oncogene during Tumor ... · in T24 bladder carcinoma cells (18). Isolated ras oncogenes were found to induce transformation and tumorigenicity

(CANCER RESEARCH 48, 4941-4953, September 1, 1988]

Clonal Diversity of the Kirsten-ras Oncogene during Tumor Progression in AthymicNude Mice: Mechanisms of Amplification and Rearrangement1

Robert Radinsky2, Paul M. Kraemer, Max R. Proffitt, and Lloyd A. Gulp

Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Â¡R.R., L. A. C.J; Los Alamos NationalLaboratory, Experimental Pathology Group LS-4, Los Alamos, New Mexico 87545 [P. M. K.J; and Department of immunology and Cancer Research, Cleveland ClinicFoundation, Cleveland, Ohio 44106 [M. R. P.]

ABSTRACT

Single-cell clones from primary and lung metastatic tumors have beenevaluated for the state of the viral-Kirsten-ras oncogene (v-Ki-ru.v) bySouthern blot analysis after injection of Kirsten sarcoma virus-transformed BALB/c 3T3 cells (KiMSV, with a replication-defective provirus)

into athymic nude mice by four different injection routes. While all clonesof early-passage KiMSV cells contained an fcoRI-generated 5.3-kilobaseDNA fragment at high dosage level, most clones of late-passage cellshad lost this v-Ki-ra.v fragment or had greatly diminished levels. However,all clones of all tumors (>90 tested) obtained after injection of these late-passage cells contained a dosage of the 5.3-kilobase v-Ki-ru.v band similarto that of the early-passage KiMSV cells, suggesting either a very strongselection for v-Ki-ras-bearing cells of the early-passage type in tumorformation and/or the ability of a subset of late-passage cells to amplify

this gene to some minimal dosage level. Both flow cytometric analysesfor DNA content and quantitation of chromosomes showed that allprimary and lung metastatic tumors had more than twice the number ofchromosomes as the late-passage KiMSV cells; however, four of 80 late-

passage cells had a chromosome count in the range of tumors, consistentwith their importance in tumor generation and possibly amplification ofthe v-Ki-rÃ»Ã®-bearingchromosome. Clonal analyses of lung micrometas-tatic tumors revealed a v-Ki-ros blot pattern identical to that of the s.c.

primary tumors. However, two of five lung mÃ©tastases from the footpad(as large rapidly growing nodules) and i.v. routes had multiple copies ofv-Ki-ruv in new sites; a second injection round led to even greatercomplexity in v-Ki-ru.v patterns in clones of lung tumors. Two assayswere used to demonstrate that these new v-Ki-ru.v integrations weregenerated by superinfection with a "helper" retrovirus, not sarcomagenic

by itself in the nude mice, that led to rescue/reinfection of tumor cellswith the defective Kirsten sarcoma provimi genomeâ€”cellular transfor

mation of 3T3 or C3H10T1/2 cells and RNA dot blot analyses formedium-secreted retrovirus specific for LTR or v-Ki-nu sequences. This"helper" retrovirus could not be detected in early- or late-passage KiMSV

cells used for inoculation but could be detected in certain tissues ofnormal nude mice, demonstrating its in vivo origin. In summary, clonalanalyses of the v-Ki-ru.v oncogene in both primary and lung metastatic

tumor populations have suggested mechanisms for amplification andrearrangement of this oncogene during tumor progression in this systemand provide the first evidence for synergy between a nude mouse-borne

retrovirus in generating new insertion sites for this oncogene, makingthem more highly metastatic in the lung site.

INTRODUCTION

Tumor progression may require activation, mutation, or lossof one or more classes of genes (1, 2). At least three categoriesof genes are postulated to play roles in neoplastic processesâ€”oncogenes (3, 4), tumor suppressor (5), and modulator genes(6). Oncogenes act dominantly and transform cells in culture.One oncogene family, raÃ,is involved in transformation, tu-

Received 3/17/88; revised 5/25/88; accepted 6/3/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Research Grant CA27755 (L. A. C.) and the Departmentof Energy (P. M. K.). Case Western Reserve University is a comprehensive cancercenter supported by USPHS Grant P30CA43703, awarded by the NationalCancer Institute, Department of Health and Human Services.

2To whom requests for reprints should be addressed.

morigenicity, and malignancy in certain cell types, includingthe BALB/c 3T3 cells used here. At least four types of ras genesexist in mammals. v-Ha-ras3 and v-Ki-ras were first discoveredin the replication-defective genomes of the Harvey and Kirstenmurine sarcoma viruses (7, 8), with cellular analogs discoveredin uninfected mammalian cells (9). N-ra.v was found in human

tumor DNA as a participant in transformation (4, 10); however,N-ras is not associated with a known retrovirus. The fourth, R-rai, was isolated from mammals by its homology to c-Ha-rosDNA sequences (11). Cellular and viral analogs of ras genesencode a similar M, 21,000 protein (p21), identified as an innersurface membrane protein that binds guanine nucleotides andthat has GTPase activity (9).

Activation of ras genes to ones with transforming and tumor-inducing potential occurs by either qualitative or quantitativemechanisms. Point mutations resulting in amino acid substitutions at positions 12 or 61 in the p2l protein have beenobserved (12). This mechanism was confirmed by site-directedand random mutagenesis studies (13, 14). In addition, trans-genie mice developed tumors in the pancreas when "activated"ras genes, not normal proto-oncogenes, were under the control

of the rat I elastase regulatory element (15). Chang et al. (16)showed a second mechanism of activation by increasing thelevel of the ras proto-oncogene's product, p21, resulting in

tumorigenic conversion of NIH 3T3 cells. Transfection experiments using DNA from naturally occurring tumors have revealed "activated" c-Ki-roi in numerous carcinomas, sarcomas,and solid tumors ( 17); "activated" c-Ha-rai has also been found

in T24 bladder carcinoma cells (18). Isolated ras oncogeneswere found to induce transformation and tumorigenicity inestablished cell lines but in primary cells a second oncogenewas required (19). These data indicate that the tumorigenicphenotype can be transferred to cells using specific DNA sequences such as ras.

Studies have also suggested that the metastatic phenotypecan be transferred to nonneoplastic cells through insertion ofras oncogenes (20-27), kinase-encoding oncogenes (28), or

tumor DNA (29). There was no clear correlation between rasgene expression and metastatic competence when metastaticlesions were compared to primary tumor (30) or to the originally injected cells (23, 31). Therefore, a basal level of raÃexpression may be necessary to induce the metastatic phenotypebut is certainly not sufficient. Some caveats should be noted."Preneoplastic" cell lines such as NIH 3T3 became tumorigenic

and neoplastic 20 weeks after injection without insertion of aras oncogene (21). Kerbel et al. (32) observed that transfectionof a plasmid containing the ncomycin selectable gene (withoutraÃ)could induce some metastatic competence in recipient cellsand calcium phosphate treatment of cells, for transfection procedures, has weak transforming ability. Nevertheless, the ras

3The abbreviations used are: v-Ha-ros, viral-Harvey-riW oncogene; c-Ki-roÃ¯,cellulai KirMcn ra.vproto-oncogene; cl, clone; KiMSV, Kirsten murine sarcomavirus; LTR, long terminal repeat; MoMuLV, Moloney murine leukemia virus;Pr, primary; Met, metastatic; CHO, Chinese hamster ovary; HSR, homogeneously staining region.

4941

Research. on September 19, 2020. © 1988 American Association for Cancercancerres.aacrjournals.org Downloaded from

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CLONAL ANALYSES OF Ki-ras AND TUMOR PROGRESSION

gene is a strong signal for effecting metastatic capability. Theseresults also indicate that rax is needed for the induction and/orestablishment of the metastatic phenotype but may not beneeded for independent growth at the secondary site (23).

In light of these studies, our laboratory showed that injectionof KiMSV-transformed BALB/c 3T3 cells into athymic nudemice by four anatomical routes resulted in rapidly growingprimary tumors that metastasized to lungs; in most cases,micrometastases were observed while in several cases overt lungnodules were observed (33). Southern and mRNA blot analysesof the cellular and viral Kirsten-ros genes (c-Ki-ras and v-Ki-rax. respectively) in tissue culture-adapted primary and lungmetastatic tumor cells showed v-Ki-ros gene amplification (4-to 8-fold) and increased mRNA expression (4- to 60-fold) whencompared with the originally injected KiMSV cells. Amplification of v-Ki-ros and increased mRNA expression were comparable in many of the primary and secondary tumor cell lines.However, in two of five lung tumors from the i.v. and footpadinjection routes, Ki-ras rearrangements (extra Ki-ras hybridiz-able bands) were observed in addition to the v-Ki-ros at itsoriginal integration site. Injection of footpad lung tumor cellswith KÂ¡-/-Â«.vrearrangements into a second group of animals led

to multiple and rapidly growing lung mÃ©tastaseswith even moreKi-wv rearrangements. In contrast, injection of an i.v. lungmicrometastatic tumor cell with Ki-ra.v rearrangements led tono further rearrangements in the lung micrometastases subsequently isolated. These results were consistent with, but did notprove, the importance of amplification and elevated expressionof v-Ki-ros in tumor progression and correlation of the Ki-rasrearrangements with formation of rapidly growing metastaticlung tumors in this system (33).

The goal in this study was to analyze the state of the c-Ki-rasand v-Ki-ros genes in multiple single-cell clones of these tumorpopulations to gain insight into the mechanisms for v-Ki-rosamplification and the Ki-ras rearrangements. Tumor populations were dilution-cloned immediately after culture outgrowthand single cell clones analyzed by Southern analyses usingseveral specific probes. Chromosome quantitation and DNAcontent analyses were performed on these tumor cell populations and on the originally injected KiMSV cell population.Mechanisms for both v-Ki-ros amplification and for Ki-rasrearrangements in overt-growing metastatic lung tumors aresuggested from these studies.

MATERIALS AND METHODS

Cell Lines. BALB/c 3T3 (clone A31), Kirsten murine sarcoma virus-transformed BALB/c 3T3 (nonproducer clone k-234, referred to asKiMSV) (34-36), and C3H10T1/2 cells (37) were grown Mycoplasma-free in DME with 250 units/ml penicillin, 250 i/g/ml streptomycinsulfate, and 10% fetal bovine serum (33). Primary tumor and lungmetastatic cell lines were established as described (33). Briefly, 10*

KiMSV cells were inoculated s.c., i.v., i.m., or into the hind footpad ofBALB/c (nu/nu) athymic mice (for description of nude mouse colony,see Reference 38). Lethargic animals were sacrificed and at autopsy themajor organs examined grossly and histologically for visually overt ormicrometastases. Overt tumors were dispersed into culture with 0.25%trypsin/50 mM EDTA and serially propagated in vitro no more thansix times prior to analyses. Micrometastases were isolated by dispersingwhole lungs (other organs were negative) into tissue culture to allowovergrowth of tumor cells which routinely occurred in 1-2 weeks.

Cloning of Cells. Dilution cloning of tumor populations was carriedout following trypsinization to a concentration of one cell per 0.2 mlmedium inoculated into wells of a 96-well cluster dish. Verification ofone cell per well was performed by phase contrast microscopy. Cellswere grown into mass cultures after several passages for analyses of

their DNA. Anchorage-independent growth in media containing meth-ylcellulose (39) occurred after plating cells in 6 ml of medium containing10% fetal bovine serum, 1.45% methylcellulose (w/v), and 90% DMEat densities from IO2to 10*cells per 100-mm dish over a layer of 0.6%

agar (w/v), 10% fetal bovine serum, and 90% DME. Cultures were fedan additional 4 ml of methylcellulose medium biweekly and colonies(50-100 cells) were isolated at 2-3 weeks.

DNA Analyses. For Southern blot analyses, DNA was extracted fromIO8 cells by Dounce homogenization of the cell pellet in 9 ml of a

solution containing 100 mM NaCl, 10 mM Tris (pH 8.0), and 1 mMEDTA (33). Pronase (500 Mg/ml), NaDodSO4 (0.5% w/v), and EDTA(20 mM) were added to the homogenate for incubation at 37 Â°Cfor 3 h.

The solution was extracted with phenol/chloroform and dialyzedagainst 50 mM Tris (pH 8.0), 10 mM EDTA, and 10 mM NaCl overnightat 4Â°C.RNase A (50 Mg/ml) was added for 2 h at 37"C. The digest was

extracted with phenol and then multiple times with a phenol/chloroform solution; final dialysis was performed against 10 mM Tris (pH8.0), 1 mM EDTA overnight at 4Â°C.DNA digestions with restrictionendonucleases were conducted at 37Â°Cfor 4 h and Southern blot

analyses/hybridization performed as described (33). Filters were washedat 68'C with 30 mM NaCl, 3 mM sodium citrate (pH 7.2), 0.1%

NaDodSO4 and bands quantitated on a Shimadzu Chromato Scanner.Probes for DNA Sequences. All probes were synthesized by nick-

translation of restriction fragments using [a-32P]deoxyribonucleotide

triphosphates. DNA fragments to be labeled were purified by agarosegel electrophoresis and electroelution. Four DNA probes were used: (a)1.0-kilobase EcoKl restriction fragment (corresponding to exons 1 thru4) of the v-Ki-ros oncogene from plasmid pHiHi 3 (40), (b) 0.964-kilobase IIini\\\\~lial\ restriction fragment (specific to the LTR) fromthe plasmid pEVX containing a truncated Moloney murine leukemiavirus genome (41), (c) 1.2-kilobase Jird actin gene fragment (courtesyof Dr. T. Nilsen of this department), and (d) EcoRl-Hindlll DNAfragment representing 3'-untranslated material from a 3.4-kilobase

EcoRl DNA insert in the plasmid pY413 containing c-Ki-ras exon 4Band adjacent 3' and 5' flanking DNA (42) (graciously provided by Dr.

Donna L. George, Department of Human Genetics, University ofPennsylvania, School of Medicine, Philadelphia, PA). This last probecontaining 3'-untranslated material did not cross-react with KiMSV

viral DNA.Viral RNA Dot Blot Assay. Tissue culture media (20 ml) was har

vested at two consecutive 24-h intervals from subconfluent cells in 175-cm2 flasks. Cell debris was removed by centrifugation (10,000 x g for

0.25 h) and virus particles pelleted by ultracentrifugation through alml sucrose cushion [20% (w/v), 20 mM Tris (pH 7.4), 0.1 M NaCl] at25,000 x g for 2 h at 4Â°C.RNA was extracted from the pellet in buffer

containing 0.5% (w/v) NaDodSO4, 0.2 mg/ml proteinase K, and 10Mg/ml tRNA added as carrier. After 30 min at 50Â°C,the homogenate

was extracted with phenol/chloroform and RNA precipitated withethanol. RNA was resuspended in H2O:formaldehyde (3:1 v/v), heated15 min at 65Â°C,and diluted 1:4 and 1:16 with 16x SSC [Ix SSC =

0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)]. RNA was dot blottedonto GeneScreen and hybridized with the designated radioactivelylabeled DNA probes.

Cellular Transformation Assay. Using cellular focus formation toassay murine sarcoma virus for its ability to morphologically transformindicator cells (43), monolayers of either BALB/c 3T3 or C3H10T1/2cells were incubated with filtered (0.45-iim pores) culture media harvested from subconfluent tumor cells (as described above) for 2 h.Medium was replaced with fresh medium every 3 days. At Day 14,these monolayers were photographed by phase contrast microscopy,fixed with 95% methanol, stained with 10% (v/v) Giemsa's stain:H2O,

and photographed a second time by bright field microscopy. Molecu-larly cloned MoMuLV was purified from the medium of NIH Swiss3T3 cells (21-1S/NIH) transfected with pBR322 containing theMoMuLV genome (44) (obtained through the courtesy of Drs. W. D.Hankins and E. M. Scolnick of the NIH). The supernatant was foundto contain approximately 1 x IO6 XC plaque-forming units/ml (45)and reverse-transcriptase activity of 1.6 x 10' counts per minute/ml

(46). As a positive control, the Kirsten murine sarcoma virus wasrescued from subconfluent KiMSV cells by adding MoMuLV (at dilu-

4942



CLONAL ANALYSES OF Ki-nu AND TUMOR PROGRESSION

lions of 1:10 or 1:20 of stock titer) to the medium of a culture for 0.75h; media was replaced with fresh medium and the cells grown toconfluence. Supernatants from these cells contain both the MoMuLVand rescued KiMSV viruses as described below.

DNA Content Analysis. For DNA content, whole cells were fixed inphosphate-buffered saline/ethanol and stained in 100 MK/nil mithra-mycin in Tris buffer (pH 7.3), 20 mM MgCl2 (47). Cells were analyzedon the flow cytometer at the Los Alamos National Laboratories (48).CHO cells were used as an internal control, directly relating DNAcontent to chromosome number. Single parameter data were displayedin 256 channels as a histogram. Mithramycin-stained cells were alsoexamined by fluorescence microscopy for confirmation that a singlecell suspension existed prior to flow analysis.

Chromosome Analysis. Chromosomes were prepared by standardprocedures (38). Briefly, KiMSV and tumor cell lines were colcemid-blocked for 2 h, harvested by trypsinization, and pelleted by centrifu-gation. The pellet was re-suspended in 1 ml of medium made hypotonie

by adding 7 ml of 0.075 N KC1, cells pelleted again by centrifugation,and finally cells fixed twice in a freshly prepared 3:1 (v/v) met ham il-acetic acid solution for 10 min. The final pellet was re-suspended in

fixative, spread on cold wet slides, and air dried. Slides were stainedwith 10% (v/v) Giemsa's stain:H2O (pH 6.8) for 10 min and photomi

crographs made of >72 metaphase spreads for each cell line for chromosome quantitation.

Materials. Cluster dishes and 100-mm l'etri dishes were obtained

from Costar, Cambridge, MA; tissue culture flasks from Becton Dickinson Labware, Oxnard, CA; DME and colcemid from GIBCO, GrandIsland, NY; fetal bovine serum from Biologos, Inc., Naperville, IL;methylcellulose (400 cps), methanol, chloroform, formaldehyde, andmicroscope slides from Fisher Scientific Co., Fairlawn, NJ; Difco-Bacto agar from Difco Laboratories, Detroit, MI; proteinase K andrestriction enzymes from Boehringer Mannheim Biochemicals, Indianapolis, IN; [a-32P]deoxyribonucleotide triphosphates from Amer-

sham Corp., Arlington Heights, IL; EDTA, RNAse A, and tRNA fromSigma Chemical Co., St. Louis, MO; Pronase from Calbiochem, SanDiego, CA; GeneScreen from Du Pont-New England Nuclear, Boston,

MA; agarose (electrophoresis grade) from Bethesda Research Laboratories, Gaithersburg, MD; nitrocellulose (0.45 ftm) from Schleicher &Scimeli. Inc., Keene, NH; technical pan film 2415 and Kodak diagnosticfilm were from Eastman Kodak Co., Rochester, NY; mithramycin wasfrom Pfizer, Inc., Croton, CT.

RESULTS

Tumor Formation in Athymic Nude Mice. Injection ofKiMSV-transformed BALB/c 3T3 cells into athymic nude miceproduced rapidly growing tumors by the s.c., i.m., and footpadroutes (33). These viral Kirsten ras (v-Ki-ros)-induced primarytumors were progressive and formed lung mÃ©tastases3 to 4weeks postinjection. Histology of primary tumors revealed irregularly shaped cells indicative of fibrosarcomas (Fig. IB)quite different from lung tissue (Fig. \A). Examination of lungfields demonstrated either overt (pea size) or microfocus met-astatic tumors (Fig. 1, C and D). Microfoci were confirmed asbeing tumor derived by adapting these lungs to in vitro cellculture with resultant tumor cell overgrowth within 1 to 2 weeks(33; see below). Specific tumor cells were also injected into asecond group of nude mice (round II), and the resulting lungmÃ©tastasesisolated for in vitro growth (33). Histology of secondround lungs revealed either overt or microfocus mÃ©tastases(data not shown).

Amplification and Rearrangement of the Viral-Kirsten-roi Oncogene. It has previously been shown that the v-Ki-ra.voncogenemay be amplified in these tumors and Ki-ra.v gene sequencesrearranged in select cases (33). Using DNA blot analysis, theuntransformed BALB/c 3T3 cells contain only the endogenousc-Ki-ras proto-oncogene (11.5-, 8.5-, 1.5-, and 0.7-kilobase

EcoRl restriction fragments in Fig. 2, lane A). In addition, the

KiMSV parental cells used for injection (passage 22) containeda 5.3-kilobase EcoRl DNA restriction fragment indicative ofthe unique KiMSV (v-Ki-ros containing) provirus (Fig. 2, laneB) (33). All tumor cell populations (>30 examined) containedthis 5.3-kilobase EcoRl restriction fragment providing directevidence that tumors induced in vivo and adapted to in vitrogrowth were derived from the originally injected KiMSV population (Fig. 2, lanes F-K). Densitometric quantitation of aratio of the intensities of the 5.3-kilobase v-Ki-ros and the 1.5-kilobase c-Ki-ras bands from primary or secondary tumor cellsrevealed a 4- to 8-fold amplification of v-Ki-roj when comparedwith the originally injected KiMSV cells (Fig. 2, compare lanesF-K with lane B) (33). Ki-ros rearrangements (additional Ki-ras hybridizable bands) were observed in lung metastatic cellpopulations from the i.v. and footpad injection routes (Fig. 2,arrows, lanes H and /, respectively), and upon a second roundof tumor formation even further rearrangements were observedfor lung metastatic tumor cells originally from the footpadinjection route (Fig. 2, arrows, lane K) (33). Molecular basesfor v-Ki-ros amplification and Ki-ras rearrangements were investigated further by analyzing many clones from these tumorpopulations for the state of the c-Ki-ras and v-Ki-ros genes.

DNA blot analysis compared the injected KiMSV cell population (passage 22) with the same cells at passage 6, soon aftertheir original virus transformation and cloning. In high passagecells, a substantial decrease in the intensity of the 5.3-kilobasev-Ki-roj DNA fragment (using the 1.5-kilobase c-Ki-ros DNAfragment as an internal control for DNA concentration) wasobserved relative to the early-passage cells (Fig. 2, comparelanes B and D). This result indicates that during longterm invitro growth dosage of the v-Ki-roj gene is reduced. Experiments

have shown that when passage 22 KiMSV cells are injectedinto athymic nude mice, the intensity of the 5.3-kilobase v-Ki-ras DNA fragment in all primary tumors and lung metastatictumors is comparable to passage 6 cells and not with the lowlevel of intensity in passage 22 cells (33).

Further study of Ki-ros genes in KiMSV cells (passage 6 or22) was undertaken by dilution cloning and subsequent Southern blot analyses. Ten single-cell clones at passage 6 yielded anidentical DNA restriction enzyme fragment pattern and intensity of the 5.3-kilobase v-Ki-ros DNA fragment when comparedto the total uncloned population (Fig. 2, compare lanes D and/:'); this intensity was comparable to the same band in the

primary tumors and lung metastatic cells (>30 analyzed) (Fig.2, compare lane E with lanes F-K). In contrast, most single-cell clones from passage 22 cells showed the absence (or severelyreduced intensity) of the 5.3-kilobase \-Ki-ras fragment ascompared with the total population or with passage 6 clones(Fig. 2, compare lanes C with B and E). Therefore, only a smallsubpopulation of passage 22 cells have retained the 5.3-kilo-base-characterized v-Ki-ros copy found in all passage 6 clones,revealing the significance of v-Ki-ros dosage in the formationof tumors in this system. There are two possible origins of the5.3-kilobase v-Ki-ras dosage in these tumors: (a) very strongselection for a minor subpopulation of passage 22 cells thathave retained the level of v-Ki-ros observed in passage 6 cells;(h) alternatively, a subpopulation of cells upon in vivo pressuremay be able to amplify the v-Ki-ros gene to some minimumlevel required for tumor progression.

Chromosome/I)NA Content Analyses of Tumor Cell Populations. That the v-Ki-roj gene is "amplified" from 2- to 8-fold in

both primary and lung metastatic tumor cells compared withuncloned passage 22 cells is consistent with a mechanism inwhich duplications of a specific chromosome containing the

4943



CLONAL ANALYSES OF Kj-ras AND TUMOR PROGRESSION

a&-,â€¢:--,â€¢.1P* â€¢â€¢*/- " ' s?.-â€¢-sÂ«.T; _-p â€¢%

Fig. 1. HistolÃ³gica! analysis of primary tumors and tumor-bearing lungs. KiMSV cells were inoculated into athymic nude mice by the indicated routes, and at 3to 4 weeks postinjection the animals were sacrificed for histology of primary tumor and lung tissue. Primary tumor and lung tissue were thin sectioned, lionate >\ylinand eosin stained, and histologically examined. .-(.normal mouse lung Held (X175); />',KiMSV cell-induced primary tumor resulting from an s.c. route of injection(xllS); C, KiMSV cell-induced lung metastasis resulting from i.v. route of injection (xl7S); /', KiMSV cell-induced lung micrometastasis resulting from the s.c.route of injection (X17S). Magnifications are given in parentheses.

B D E H K

Fig. 1. Southern blot analyses of tumor celllines. Genomic DNA (25 ^g per lane) was cutwith restriction endonuclease EcoRl andprobed with a 1-kilobase DNA fragment ofpHiHi 3 (40). This probe hybridizes with bothv-Ki-ros and c-Ki-ras sequences (33). A v-Ki-ras DNA fragment of 5.3-kilobases and c-Ki-ros-specific bands of 11.5, 8.5, 1.5, and -0.7kilobases are expected when probed withpHiHi 3 (33, 40). DNA fragment sizes wereestimated by using //indlll-digested A DNAand //ad 11-digested 0X174 phage DNA.Lanes: A, BALB/c 3T3 cells; B, originally injected KiMSV cells (passage 22); C, KiMSVpassage 22 single cell clone (II III); /).KiMSV cells (passage 6); E, KiMSV passage6 single cell clone (B-3); F, Pr (primary) S.c.r1; G, Met (secondary) s.c.i-1; H, Met i.v.i-1; /,Pr Ftpd,-3; J, Met Ftpd,-lT; K, Met Ftpd,-lT

â€¢i.v.u-lBT [subscript I and II indicate cellsfrom round I or II injection schemes, respectively (33)]. Note that data from several different gels were combined to make this compositefigure. Arrows, additional rearranged ki-ra.vbands in three cases. The size in kilobases ofDNA fragments is shown at the right with <â€¢=cellular-Ki-rof and v = viral-Ki-ros.

â€¢II.5C8.5c

'5.3v

â€¢l.5c

â€¢0.7c

KiMSV provirus, a chromosomal segment containing the pro-virus, or a large segment of DNA (> 1,000 kilobases) containingv-Ki-ros is disproportionately replicated (i.e., more than onceper mitotic event) (49). This possibility was investigated byquantitating chromosome number in metaphase cells from spe

cific tumor populations. Tumor cells contained at least a twofold increase in chromosome number compared to the originallyinjected KiMSV cells at passage 22 (Table 1 and Fig. 3). DNAcontent analysis by flow cytometry confirmed this and showeda twofold increase in modal chromosome number in tumor

4944



CLONAL ANALYSES OF Ki-raj AND TUMOR PROGRESSION

Table 1 Quantitation of chromosomenumberFlow

cytometricDNA contentanalysis"Tumorcell

lineKiMSV

(parental)CHOPr

s.c.i-1CHOMetFtpdrlTCHOMet

Ftpdi-1->i.v.i,-lBTCHOGÃ¬

mode100.1375.27184.6065.79217.6678.84192.2266.65Ratio1.332.802.762.88Expectedmodalchromosomeno.53112110115Metaphasespreads*,averagechromosomeno.52.5

Â±18.9(n=80)109.7

Â±19.3(n=72)104.8

Â±18.8(n=76)102.1

Â±12.8(n= 81)

" Mithramycin-stained cells were analyzed for DNA content using flow cytom-

etry (47, 48). The expected modal chromosome number was calculated from theDNA content data using CHO cells as a control for diploid mammalian DNAcontent. CHO cells contain 21 chromosomes per cell, but nearly normal DNAcontent. The ratio was calculated by dividing the G> mode of the test cells by theG i mode of the CHO cells; this value was then multiplied by 40 (the normalmurine chromosome value) resulting in the expected modal chromosome number.

* Cells were colcemid blocked in metaphase. Hypotonie cells were then fixed

and spread onto glass coverslips (38). Chromosomes were quantitated fromphotomicrographs of individual metaphase cells. The average chromosome number and standard deviation are given, n â€”number of metaphase cells analyzed.

populations as compared to passage 22 KiMSV cells (Table 1).However, four of 80 metaphase cells analyzed from passage 22KiMSV cells contained elevated chromosome numbers 80-107,comparable with the number observed in tumor cells (Fig. 3,compare A with B-D). A minor number of such cells in apopulation could easily be missed during Southern blot analysisoften randomly chosen clones. Comparison among PrS.c.i-1,Met Ftpdi-lT, and Met Ftpdi-lT â€”Â»i.v.n-lBT tumor populations showed similar chromosome numbers and DNA content(Table 1), consistent with the importance of elevated chromosome number for competence in tumor progression in thissystem. Chromosome histograms were also similar, except formetastatic populations containing Ki-ra.v rearrangements (Fig.2, lanes J and K); at least two cells were present with threetimes the chromosome number of the originally injectedKiMSV cells (Fig. 3, compare B with C and D). These resultsare consistent with a chromosome-based mechanism for twofold v-Ki-fu.v amplification, involving in vivo selection for cellswith increased chromosome number (see discussion); however,such a mechanism may not explain the >8-fold "amplification"unless the v-Ki-ras bearing chromosome or DNA segment werespecifically amplified.

Clonal Analysis of Ki-rus in Tumor Populations. These experiments led to analyses of the relative homogeneity of the v-Ki-ras oncogene in the tumor populations from nude mice, usingmany single cell clones. Southern blot analysis is estimated tobe sensitive enough to detect a unique clone at 5% of the totalcell population during analysis of the DNA from a heterogeneous population (50). Therefore, a subpopulation of cells witha unique genotypic marker and comprising less than 5% of thetotal population may be masked. The Ki-ros restriction enzymefragment pattern (including KÃ•-/VÃ•.Srearrangements) was usedas a genotypic marker to test for microheterogeneity in thestate of v-Ki-ras and/or c-Ki-ras in tumor populations whenprimary and metastatic lung tumors were dilution cloned. Inaddition, if host cell contamination exists in tumor populations,then clonal DNA blot analysis would identify a sizable level ofcontamination with host cells, since they would not contain av-Ki-ras-specific band. Ninety clones (ten single-cell clonesfrom nine different primary or secondary tumor populations,

AKiMSV-BALB/c3T3

j_ _LL35 45 55 65 75 85 95 105 115125 135

BPrS.c.j-l

O

Lu

OLuer

85 95 105 115 125 135 145 155 165 175

CHROMOSOME NUMBERFig. 3. Chromosome frequency proli losof KiMSV BALB/c 3T3 (late passage),

primary tumor, and lung metastatic cell populations. Cell populations werecolcemid-blocked in metaphase (38); chromosomes quantitated per cell andplotted versus frequency of cells containing that number of chromosomes (seeTable I). A, KiMSV-BALB/c 3T3 (passage 29); B, Pr S.c.,-1; C, Met Ftpd,-lT;D, Met FtpdrlT-Â» i.v.,,-lBT.

only three sets of which are shown in Fig. 4) showed no evidenceof host cell contamination; all clones contained the 5.3-kilobasev-Ki-ros EcoRl generated DNA fragment (Fig. 4, A-C). DNAblot analysis using the restriction enzyme Xbal confirmed theseresults.

Analysis of single cell clones from primary and lung metastatic tumors may also uncover subsets of cells selected forduring tumor progression. Ten clones from an s.c. primarytumor (PrS.c.i-1) showed a DNA blot pattern analogous to thewhole population (data not shown). In addition, a lung metastatic population (Met S.c.i-1) derived from this primary s.c.tumor showed eight single-cell clones with the same DNA blotpattern and v-Ki-ros levels as the total population, or PrS.c.i-1cells from which the tumor was derived (compare Fig. 4A cloneswith Fig. 2, lanes G and F, respectively). These results indicate

4945




Fig. 4. Southern blot analysis of single cellclones from tumor cell populations..-(, the MetS.c.i-1 tumor cell population (Fig. 2, lane G)was dilution cloned and Southern blot analysiswas performed on eight single cell clones (aftertheir outgrowth into mass cultures). GenomicDNA was cut with the restriction endonucleaseEcoRl and probed with pHiHi 3. Conditionswere identical to Fig. 2. /(. eight single cellclones are shown from the Met i.V., I cellpopulation (Fig. 2, lane H) analyzed by Southern blot analyses. Conditions were identical toA. Arrowhead, additional 8.3 kilobase Ki-rav-specific band. C, Southern blot analyses offcoRI-digested DNA from eight single clonesfrom the Met FtpdrlT tumor cell population(Fig. 2, lane J). Conditions were identical to.I. The size in kilobases of DNA fragments isshown at the right with <â€¢= cellular-Ki-nu andv = viral-Ki-ras.

A MetS.c.,-1

Cll CI2 CI3 CI4 CIS CI6 CI7 CIS

B. Met lv.,-1

Cll CI2CI3CI4 CI5CI6CI7CI8

C. Met Ftpd ,-IT

Cll CI2 CI3CI4 CIS CI6 CI7 \f

i -Â«-use

' -Â«-53v

â€¢fe

the stability of v-Ki-ros during tumor progression via the s.c.inoculation route.

Clonal analyses of two different lung metastatic tumors withKi-ra.vrearrangements demonstrate different results. In the firstcase, eight clones from an i.v. experimental lung tumor (Meti.v.i-1) containing a more complex DNA restriction enzymefragment pattern (i.e., with extra 8.3-kilobase and 4.2-kilobaseKi-ras bands; Fig. 2, lane H) yielded the same Ki-ras pattern asthe total population (compare Fig. 4B with Fig. 2, lane H).However, one of these clones contained even more Ki-rasrearrangements (Fig. 4B, cl 3). No clone was found without theki-n/v rearrangements found in the original Met i.v.,-1 population. Therefore, this lung tumor cell may be derived from avery minor cell type in the KiMSV cell population (passage 22)or genotypic changes are occurring during in vivoselection thatconfer an advantage to these cells during metastasis.

In the second case, Met FtpdrlT tumor cells [a metastaticlung nodule isolated from the footpad route with multiple Ki-ras rearrangements (Fig. 2, lane J)] generated eight single-cellclones with multiple Ki-ras DNA fragment patterns (Fig. 4C).Two clones (Fig. 4C, cl 3 and cl 8) exhibited a pattern similarto the total Met Ftpdi-lT cell population (Fig. 2, lane J) andone clone (Fig. 4C, cl 7) had a similar Ki-ras pattern as theoriginally injected KiMSV cells (Fig. 2, lane B). These resultssuggest heterogeneity in the state of the Ki-ra.vgenes in MetFtpdi-lT cells. Interestingly, in a footpad primary tumor(PrFtpdi-3, Fig. 2, lane I) and in a s.c. tail tumor (Pr i.v.i-1),no Ki-ras rearrangements were present from the DNA isolatedfrom the total populations; in contrast, individual clonesshowed multiple patterns of Ki-ras rearrangements (see Table2). It should be noted that all clones contained the original v-Ki-ras 5.3-kilobase EcoRl DNA fragment. These data indicatethat DNA blot analysis on the total tumor population can maskgenotypic diversity in small subpopulations and that the molecular basis for these Ki-ras rearrangements must be deciphered.

Several experiments suggest that these clonal data are a validreflection of the situation in vivo.Ninety individual clones fromtumor populations were found to contain the 5.3-kilobase v-Ki-ras EcoRl DNA fragment; no clone was ever missing this band,indicating that the KiMSV provirus is stably integrated in thissite (confirmed using the restriction enzyme Xbal) (Fig. 4, anddata not shown). Cloned cell lines, as well as the original tumorpopulations, were grown longterm in tissue culture to assessthe stability of the rearrangements and DNA fragment patterns.DNA blot analyses on early passage (passage 6) or late passagecells (>passage 18) and clones showed no alterations in Ki-rasblot patterns as a consequence of in vitro growth (data notshown); therefore, selection pressures in vivo during tumor

progression are very different than any selective pressures invitro.

To test for genotypic instability in tumor cells and in theoriginally injected KiMSV cells, various in vitro selection pressures were used. If alterations were due to dilution cloningitself, rearrangements in the PrS.c.i-1 and Met S.c.i-1 cellpopulations would be expected and none were found (Fig. 4A).Methylcellulose-based cloning (to select for cells with anchorage independent growth) of the original KiMSV cells (bothpassage 6 and passage 22), Pr Ftpdj-3, and Met Ftpdi-lT cellsshowed no differences in Ki-ras fragment pattern as comparedwith the results derived from the dilution cloning experimentsdescribed above (data not shown).

Molecular Bases for Ki-ras Rearrangements. Ki-ras rearrangements in lung tumor populations and their single-cellclones might result from one of several mechanisms. Replication-defective KiMSV provirus may be rescued by a helperretrovirus present in vivoor in the originally injected cells. NewKi-ras hybridizable bands in the DNA blot analysis would thenrepresent new proviral integrations. Experimental evidenceproving this mechanism relies on the fact that these tumor cellpopulations would produce retrovirus. Alternatively, chromosome-based rearrangements could be invoked, possibly involving c-Ki-rosand/or v-Ki-ros.c-Ki-ros rearrangements have beenruled out in this system using a DNA probe specific to the 3'-untranslated DNA sequences in the c-Ki-ros gene; Southernblot analyses of round I and II tumor cells as well as multiplesingle-cell clones showed no alterations as compared to theoriginally injected KiMSV cells or BALB/c 3T3 cells (data notshown). Therefore, the new insertions reactive with the pHiHi3 probe must be v-Ki-ros, consistent with a viral rescue/reinte-gration mechanism.

To test this further, a cellular transformation assay was usedto assay for a helper retrovirus. Helper virus would lead toKiMSV virions being produced by tumor cell populations containing Ki-ras rearrangements; hence, these virions should becompetent for transforming mouse fibroblasts in vitro. Mono-layers of untransformed BALB/c 3T3 or C3H10T1/2 cells wereincubated with filtered medium harvested from subcontinenttumor cells (with or without Ki-ras rearrangements) for 2 h;monolayers were subsequently grown for 14 days with a mediumchange every third day. The extent of morphological transformation was scored from one to four plus as compared to positiveand negative controls, using media from BALB/c 3T3 orC3H10T1/2 cells as the negative control (Fig. 5A; data notshown for the C3H10T1/2 cells). The positive control wasmedium from KiMSV-BALB/c 3T3 (passage 22) cells in whichin vitro rescue of the KiMSV with purified Moloney murine

4946




leukemia virus was performed (Fig. 55). MoMuLV virus wasunable to transform the BALB/c 3T3 or C3H10T1/2 by itself(data not shown). A direct correlation with the DNA blotanalyses was observedâ€”i.e., all tumor cells with Ki-ros rearrangements elicited positive morphological transformation(Fig. 5, D-F), and cells without Ki-ras rearrangements showedresults identical to negative controls, as well as to mediumtaken from the originally injected KiMSV cells (Fig. SO, datanot shown). Culture medium from KiMSV cells at passage 6or 22 yielded no morphological transformation of either BALB/c 3T3 or C3H10T1/2 cells, indicating that the originally injected cells do not harbor a retrovirus which rescues the v-Ki-

ras oncogene. Table 2 (column b) summarizes these results,which support the proposal that rescued infectious KiMSV

virions are present in the supernatants of tumor cells with Ki-ras rearrangements but not in cells without rearrangements.

These findings were confirmed using a molecular biologicalapproach. RNA was extracted from putative viral particlesisolated from tumor cell culture media for testing with hybridization probes in a dot blot assay. The first probe, pHiHi 3specific to Ki-ras, hybridizes with only KiMSV RNA containingthe v-Ki-ros oncogene. A second probe, specific to the LTR

sequences of retroviruses, hybridizes to both helper and KiMSVretroviruses; however, this probe also yields reactivity withmouse DNA, demonstrating LTR-like sequences as a "background." The third probe, specific to actin, was used to control

for contamination in the viral RNA preparations with cellularDNA or RNA as a result of possible cell lysis; negligible

Ã¬&CÂ»'' t.1 â€¢'Â»..*â€¢> *

---

.

Fig. S. Cellular transformation to detect transforming retrovirus in tissue culture medium from tumor cell lines. A monolayer of BALB/c 3T3 cells was incubatedwith filtered (0.45-*tm pore size) tissue culture medium harvested from subconfluent tumor cells for 2 h. The medium was removed and replaced with fresh mediumevery three days. Fourteen days later, the cells were lived, stained with 10% Giemsa, and photographed under bright field microscopy. Tissue culture medium wastested from A, BALB/c 3T3 cells; B, KiMSV cells in which the KiMSV virus was rescued by adding purified MoMuLV; C, Pr s.c.i-1 tumor cell line (see Fig. 2, laneF); D, Met i.v.i-1 micrometastatic cell line with Ki-roi rearrangements (see Fig. 2, lane H); E, Pr Ftpdi-3 tumor cell line (see Fig. 2, lane l); F, Met Ftpdi-lT tumorcell line containing Ki-nu rearrangements (Fig. 2. lane J, and Fig. 4C). Irregular cells which are piling in these cultures are diagnostic of retrovirus-inducedtransformation.

4947




hybridization occurred with this actin-specific probe (data not

shown).As shown in Fig. 6, BALB/c 3T3 (negative control) cells

were negative with both Ki-ras and LTR-specific probes (Fig.6, A and B, lane A) and the rescued KiMSV cells (positivecontrol) exhibited extensive hybridization with both probes(Fig. 6, A and B, lane B). KiMSV cells at passages 26 or 6showed minimal hybridization with either probe (Fig. 6, A andB, lanes C and D), except for some reactivity with the LTRprobe (Fig. 6A, lane C) reflecting cell lysis of high passage cellsand liberation of LTR cross-reactive sequences, s.c. primary ormetastatic cell populations with no Ki-ras rearrangements (Fig.2, lanes Fand G) also showed negligible reactivity (Fig. 6, lanesE and F, respectively). In contrast, tumor cells with Ki-rasrearrangementsâ€”Met i.v.i-1, Met Ftpdi-lT, and Met Ftpdi-lTâ€”>i.v.n-lBT (Fig. 2, lanes H, J, and K, respectively)â€”showed

extensive reactivity with both probes (Fig. 6, A and B, lanes G,I, and K, respectively), indicating a direct correlation with Ki-

ras rearrangements and retrovirus production by these cells(Table 2, column a) and confirming the cellular transformationassay. These data are consistent with the premise that theKiMSV-provirus is rescued by a helper retrovirus in vivo andnot from an in vitro contaminant virus, but only via the footpadand i.v. routes of inoculation.

The origin of this helper virus could be the originally injectedKiMSV cells or the athymic nude mouse itself. Cellular transformation and viral RNA dot blot assays rule out the firstpossibility. Further evidence against helper virus in the originally injected population is evident from the s.c. route ofinoculation where no Ki-ras rearrangements and no reactivityin the two viral assays were found.

The second possibility, i.e., the athymic nude mouse harbor

ing an endogenous retrovirus, was experimentally tested. Heterozygous breeders (BALB/c/nu) in the nude mouse colony (38)are analyzed semiannually for 11 types of viruses; however,retroviruses are not included in this survey. Therefore, a viralRNA dot blot experiment was performed to determine whethernude mouse organs and tissues replicate retrovirus or whetherthe originally injected KiMSV cells "activate" production of a

latent retrovirus in nude mouse tissues. Cells from lung, spleen,and muscle tissues were incubated in culture media alone oradded to subconfluent cultures of KiMSV passage 29 or passage10 cells. If helper virus is present in tissues, it should be detectedwith hybridization probes and it might rescue the replication-defective v-Ki-ros provirus from these cells. After exposure tomouse tissues, cell lines were passaged in vitro three times toincrease viral titer. The media were then collected and analyzedas described above. The positive control (an in vitro rescue ofthe KiMSV using purified MoMuLV) showed hybridizationwith both probes (Fig. 7, A and B, lane B). Nude mouse spleentissue from two individual mice showed positive hybridizationwith the LTR-specific DNA probe (Fig. 7, lanes H and /) andminimal hybridization with the Ki-ras specific probe, as expected, since nude mouse tissue should not normally contain atransforming retrovirus harboring the v-Ki-ros oncogene (Fig.IB, lanes H and J). Lung tissue isolated from two individualanimals showed no hybridization with either probe (Fig. 7, Aand B, lanes I and K, respectively). Analysis of tissue culturemedium from the originally injected KiMSV cell line at passage22 cocultured with spleen or lung tissue showed some hybridization with the LTR-specific DNA probe, but no hybridizationwith the Ki-ros-specific probe (Fig. 7, A and B, lanes E and F).Similarly, medium from KiMSV-BALB/c 3T3 passage 10 cellsincubated with these same tissues showed a very small amount

A. LTRA B C D E F G H I J K

Fig. 6. RNA dot blot analyses of tumor cellline culture media. Forty milliliters of cell culture medium was harvested from subconfluentcell lines. Cell debris was removed by centrif-ugation, viral particles pelleted by ultracentrif-ugation, and RNA extracted ("Materials andMethods"). The viral RNA was resuspendedin H2O:formaldehyde (3:1), denatured at 65Â°C,

and diluted 1:4 and 1:16 as indicated. TheRNA was dot blotted on GeneScreen and analyzed using two DNA fragments as probes. A,a DNA fragment (0.964 kilobase) specific tothe LTR sequences of retroviruses (41) wasutilized to detect both helper and KiMSV retroviruses. B, pHiHi 3 (40), a 1-kilobase probespecific to Ki-ras sequences, was utilized because it will only hybridize to KiMSV retro-viral RNA. Cell lines from which culture medium was analyzed are indicated in lanes: A,BALB/c 3T3; B, KiMSV-BALB/c 3T3 cells inwhich KiMSV virus was rescued using purifiedMoMuLV as a positive control; C, originallyinjected KiMSV cells (passage 26); D, KiMSVcells (passage 6); E, Pr s.c.i-1; F, Met s.c.i-1;G, Met i.v.i-1 (Ki-ras rearrangements observed,Fig. 2, lane H); H, Pr Ftpdi-3 (Fig. 2, lane /);/, Met Ftpdi-lT with Ki-ras rearrangements(Fig. 2, lane J, and Fig. 4C); /, Met Ftpd,-2T;K, Met Ftpdi-lT-Â» i.v.n-1 BT (contains furtherKi-ras rearrangements, Fig. 2, lane K).

1:16

B.A BCDEFGH I J

1:4

4948



CLONAL ANALYSESOF Ki-raj AND TUMOR PROGRESSION

Table 2 Summary of analyses on the tumor cell populations and their single-cell clones

CelllineBALB/c

3T3BALB/c3T3KiMSV

p6cellsKiMSVp22 parentalcellsPrs.c.,-1Met

s.c.i-1KiMSVp30^ 1:20

KiMSV p30^1:10KiMSVp29^1:10Pr

i.v.i-1Meti.v.,-1PrFtpd,-3MetFtpdrlTMetFtpdi-2MetFtpd,-!â€”i.v.,,-lBTMet

Ftpdpl -Â»i.v.,,-lATMetFtpdi-1 -Â»ÃŒ.V.H-3BTViral

RNA" Cellulardot blot transformation*

assay assay Southern bloton totalceilLTR

ras BALB/c 10TÂ»population+- -Cellular-

- - -Cellularâ€”â€” â€” â€”ParentalÂ±â€” Â± -Parentalâ€”+ â€” â€”Parentalâ€”â€” + â€”Parental++++

+++ ++++ ++++ Rearrangements++++ +++ ++++ ++++Rearrangements++++

+++ ++++ ++++Rearrangements+++++++ + +++Parental++++++ ++ +Rearrangements++++++ ++++ +++Parental+++

++++ + ++++RearrangementsNDNDParentalâ€¢f

++++ ++ +++Rearrangements++++++ ++++ ++++Rearrangementsâ€”

++ + + RearrangementsSouthern

blot*

on single cellclonalpopulationsND'NDSee

"Results"See"Results"ClonalClonalND

NDNDMulticlonalMulticlonalMulticlonalMulticlonalMulticlonalNDNDND

Â°Viral RNA dot blot assay using either the LTR sequence-specific probe or the Ki-ros-specific probe. See Fig. 6 for details. Intensity of hybridization was scoredfrom 1+ = least positive to 4+ = most positive, as compared to negative and positive controls. -, no hybridization.

* Cellular transformation assay using either BALB/c 3T3 or C3H KIT., cells as described in Fig. 5. Morphological transformation was scored from 1+ = leastposition to 4+ = most positive, based on comparison with positive and negative controls (see Fig. S). -, no morphological transformation.

' Southern blot analyses of cell populations for Ki-ros DNA sequences using conditions as described in Fig. 2, legend. "Cellular" refers to only the expected c-Ki-ras DNA fragments (see Fig. 2). "Parental" refers to a ki rav DNA fragment pattern containing both the expected c-Ki-ros DNA fragments and v-Ki-ros DNAfragments (see Fig. 2). "Rearrangements" refer to Ki-ros rearrangements (i.e., extra ki-rav hybridizable bands) as observed upon Southern analyses.

Southern blot analyses of Ki-ras sequences in single-cell clonal populations via dilution cloning (see Fig. 4). "Clonal" denotes all 10 clones have an identicalfcoRI restriction enzyme DNA fragment pattern as the total population. "Multiclonal" denotes at least one clone having a different DNA restriction enzyme Ki-ras

fragment pattern as compared to the parental uncloned tumor population.' ND, no data obtained.1In vitro proviral rescue.

A. LTR

Fig. 7. RNA dot blot analysis of in vitrocultured nude mouse tissues cocultured withKiMSV 3T3 cells. The indicated nude mouseorgan or tissue was isolated sterilely andminced. This organ was subsequently culturedalone or cocultured with KiMSV BALB/c 3T3cells for 1 week. The cells were then passagedthree times prior to harvesting their culturemedium as described in Fig. 6. Viral RNAisolation from virions in cell culture media andprobes utilized were identical to Fig. 6, A andB. Tissue culture medium was collected andanalyzed from the cell lines indicated in lanes:A, KiMSV cells (passage 10); B, KiMSV cellsin which the KiMSV virions were rescued withpurified MoMuLV, positive control; C,KiMSV cells (passage 11) cocultured withnude mouse 1 spleen tissue; D, KiMSV cells(passage 10) cocultured with nude mouse 1lung tissue; E, KiMSV cells (passage 29) cocultured with nude mouse 1 spleen tissue; /â€¢'.

KiMSV cells (passage 29) cocultured withnude mouse 1 lung tissue; G, nude mouse 2muscle tissue; //, nude mouse 5 spleen tissue;/, nude mouse 5 lung tissue; J. nude mouse 6spleen tissue; K, nude mouse 6 lung tissue.

A

1:1

1:4

1:16

CDEFGHIJK

*

B.ABCD EFGH I JK

1:4

1:16

of LTR-specific hybridization and no Ki-ras-specific hybridization (Fig. 7, A and B, lanes C and D). These results suggesthelper retrovirus may be localized to spleen tissue in someindividual nude mice and activation of endogenous retrovirusproduction is not apparent from this analysis. Table 2 summarizes all of these results taken together.

DISCUSSION

Injection of KiMSV-transformed BALB/c 3T3 cells intoathymic nude mice via four routes showed rapidly growingprimary tumors with subsequent overt lung mÃ©tastasesin a fewcases or lung micrometastases in most cases (33). Southern andNorthern blot analyses showed that these tumor cells had

4949




amplified the v-Ki-ros DNA sequence from two to 8-fold andincreased mRNA expression from 4- to 60-fold (33). Furthermore, in two of five lung mÃ©tastasesfrom the i.v. and footpadroutes, Ki-ras rearrangements were observed in the DNA blots(Fig. 2) (33). Their origin as v-Ki-ros, rather than as c-Ki-ros,have now been established. The mechanisms of the v-Ki-ros"amplification" and rearrangements have been further eluci

dated in the analyses of cloned populations.That the injected KiMSV cells at passage 22 exhibited a

reduced intensity of the 5.3-kilobase v-Ki-ros band relative toc-Ki-ros when compared with the passage 6 population suggested that under tissue culture conditions v-Ki-rav gene dosagewas reduced. Southern blot analyses of single cell clones fromthese populations indicated that all clones from the passage 6population were homogeneous (identical to the total population); whereas, most clones from passage 22 population wereeither missing v-Ki-roj or had a drastically reduced level. Alltumors studied here (>30 independent isolates) had a level ofthe 5.3-kilobase v-Ki-ros DNA fragment comparable to thatobserved in passage 6 cells. This evidence is consistent with alow-frequency cell in the passage 22 population, retaining thedosage of the 5.3-kilobase v-Ki-ros band from passage 6 cellsand having selective advantage for forming tumors by all fourinjection routes. However, it cannot be excluded that amplification of v-Ki-rw.vis occurring by multiple duplications of theentire chromosome or segments of the chromosome carryingthis gene. In this regard, in situ hybridization to determine thenumber of v-Ki-roj-bearing chromosomes should be informative. Also, transfection of BALB/c 3T3 cells with varyingdosages of Ki-ras utilizing different promoters should be informative in tumor progression studies.

Heterogeneity in cell populations after growth on tissueculture substrata with respect to chromosome properties (51)or in agar suspension with respect to morphology, colonyformation, tumor formation potential in nude mice (52), drugsensitivity, metastatic potential, and type IV collagenolyticactivity (53) has been well documented. In other retroviralsystems, prolonged growth in culture of cells containing a singlecopy of provirus yielded a high proportion of subclones exhibiting 10-100-fold reduction in viral RNA and protein withouta demonstrable change in structure at the DNA level of theprovirus (54). Therefore, under relatively nonselective conditions in culture phenotypic and genotypic drift occur in tumorpopulations, as confirmed in studies described here comparingpassage 6 or passage 22 KiMSV cells with the tumor populations. Amplified DNA as a HSR or as double minute bodiescauses a cell to grow more slowlyâ€”i.e., a mixed population ofcells containing an amplification as an HSR may be overgrownby wild-type cells, even if the amplification is quite stable insubclones (49). This mechanism may explain the loss of v-Ki-nis dosage in the passage 22 KiMSV cells because the singlecell clones without v-Ki-ros or very low levels of it overgrewcells with the amplification.

These results stand in direct contrast to the results foundafter in vivo selection. All tumor populations and >90 single-cell clones contained increased v-Ki-ros gene dosage and mRNAexpression at a level characteristic of passage 6 KiMSV cellsand not the injected passage 22 cells. The rate of tumor progression in this system may depend on a minimum level of v-Ki-ras as necessary/sufficient for these complex processes tooccur in the animal (1). At least two possibilities exist forselection of a cell subpopulation from the KiMSV passage 22cells with the required level of v-Ki-ros: (a) ability to easilyamplify this gene under in vivo pressure; or (/>)selection for a

cell subpopulation with similar v-Ki-ros dosage as found in thepassage 6 KiMSV cell population or their single-cell clones.Evidence presented in this study is more consistent with thelatter mechanism, although the former cannot be rigorouslyruled out. It is intriguing as to which phenotypic changes areconferred on the cell via the activity of the p21 protein codedfor by the essential level of the v-Ki-ras gene.

That malignant competence can be established through amechanism of expanded gene dosage has been reviewed (55)and, therefore, selection for cells with the ability to easilyamplify v-Ki-ros is conceivable. Gene selective amplification formethotrexate or other drug resistance is known (49). Such amechanism for v-Ki-rt/.vamplification was analyzed to a limitedextent in our study. Flow cytometric DNA analyses and quan-titation of chromosome number of the injected KiMSV cells,as well as of various tumors, revealed at least a twofold increasein modal chromosome number for all tumors (Table 1). Increased aneuploidy and DNA content correlate with greatermalignancy (1, 55, 56). Four of eighty metaphase cells from theinjected KiMSV population contained a twofold increase inchromosome number and may provide the cell subpopulationthat yields all tumors in this system; perhaps, this same sub-population also bears the passage 6 level of v-Ki-ros (whichcould not be easily observed in Southern blot analyses of sucha low frequency subpopulation).

Precedence exists for selection of cells with a high chromosome level. In vitro, the emergence of permanent Chinesehamster cell lines from a large number of nascent stem cellsshowed the permanent stem lines to contain excess 3q chromosomal material, providing them with a competitive growthadvantage early on (57). In vivo, mutant j8-actin gene transfec-tant strains generated cell subpopulations with an ability toform tumors based on their elevated rate of mutant /8-actinsynthesis (58). Selection for cells having alterations in modalchromosome number or pattern has been observed when metastatic cell populations were compared with the primary tumorfrom which they were derived (59-61). Increased chromosomenumber and possibly increased v-Ki-ros gene dosage may confera competitive invasion/growth advantage in tumor formationin this system, as reviewed extensively (1,2, 62).

Little study has been devoted to the state of oncogenes insingle-cell clones of various tumors and such analyses have beenparticularly rewarding in this system. Nowell, Fidler, and Ni-colson have stated that tumors and their metastatic derivativesmay be initiated with single cells but that genetic homogeneitydiverges into a heterogeneous population (1, 63). Southern blotanalysis has been used to study the clonal composition oftumors containing distinct genetic markers, such as restrictionfragment length polymorphisms of X-chromosome genes (64),methylation patterns (65), and transfected plasmids containingbacterial genes (50). Such analyses could detect a unique sub-population present at approximately 5% of the total population(50). Therefore, we chose to study the clonal state of v-Ki-rosin several primary and lung metastatic tumors because Southernanalysis of the total population could mask small subpopula-tions present at <5% of the total population. Using the Ki-rasEcoRl restriction enzyme fragment pattern (including Ki-ras

rearrangements) as a genotypic marker, a stable clonal patternfor s.c. primary tumor and lung metastatic cell populations wasobserved. Single cell clones from an i.v. experimental lungmetastatic population showed nine of ten clones to contain theidentical Ki-ras DNA fragment pattern as contained in thewhole population. Southern blot analyses of clones from aprimary footpad tumor (Pr Ftpdi-3, Table 2) or a footpad overt

4950




lung metastasis showed a multiclonal pattern (i.e., different Ki-ras patterns with many new integration sites for v-Ki-r<w).Thesecell subsets were masked upon Southern analysis of the totalpopulation. In no case were the c-Ki-ra5 or 5.3-kilobase v-Ki-ras DNA fragments missing. We may postulate that overttumors were more heterogeneous in their v-Ki-ras patterns as aresult of many more cell divisions and hence diverged intomultiple subpopulations; in contrast, the micrometastases hadfew cell divisions and hence were limited in genotypic heterogeneity.

The source of the Ki-ras rearrangements from either cellularor viral Kirsten-r<w genes was differentiated, using a DNA probespecific to 3'-untranslated material of the murine c-Ki-ras gene.

In all cases new integrations of ras used the viral gene and notthe host cell gene. The origin of these v-Ki-ros rearrangementsfrom a helper retrovirus was also tested using cellular transformation and viral RNA dot blot assays. These two assays arecomplementary; the cellular assay detects a small number oftransforming retroviral virions while the molecular biologicalassay distinguishes "helper" retrovirus from KiMSV virus.

Morphological transformation and positive hybridization withKi-ras and LTR-specific probes correlated directly with tumorpopulations containing Ki-ros rearrangements and multiclonalpatterns of Ki-ras in Southern blots (see Table 2 for summary).Therefore, the Ki-ras rearrangements represent new proviralinsertions into genomic DNA. Evidence consistent with thismechanism was obtained by adding purified Moloney murineleukemia virus to KiMSV cells, thereby rescuing the KiMSVreplication-defective virus. Southern analysis of these cellsshowed additional unique Ki-ras hybridizable bands (data notshown).

Several possibilities exist for the source of the "helper"

retrovirus. First, murine retrovirus could be present in theinjected KiMSV cells. This mechanism was ruled out usingboth viral RNA dot blot and cellular transformation assays andfrom evidence that Ki-ras rearrangements were not observed inthe s.c. primary tumor or lung metastatic cell populations ortheir single cell clones. Xenotropic and ecotropic murine leukemia viruses, endogenous to athymic nude mice, can productively infect transplanted tumor cells (66-68). Reports on theinduction of these endogenous murine leukemia viruses byvarious human tumors, including oat cell carcinoma, have attributed viral inductive qualities to a specific hormone or factor(69). Chemical carcinogens, hormones, and physical agents canactivate endogenous mouse mammary tumor provirus replication (70). Therefore, in our system injection of KiMSV cellsinto nude mice harboring an endogenous retrovirus may "activate" production of the helper retrovirus in select tissues. Spleen

tissue (unlike lung tissue) cultured alone hybridized extensivelyonly with the LTR-specific probe (and not the actin probe todiscount cell lysis), indicating the presence of such a retrovirusthat could potentially superinfect KiMSV cells. Culturing nudemouse tissue alone or cocuiture with KiMSV cells yielded dataconsistent with either mechanism. A helper virus would permitreplication of the defective KiMSV in these cells and lead tomultiple reinfections with new integration sites of v-Ki-ras. Asfar as we are aware, this is the first evidence for such a syner-gistic relationship in a tumor progression system.

These data also imply a special role for v-Ki-ras in tumorprogression in this system and are consistent with studiesshowing direct correlation between ras and tumorigenesis (9).In addition to the essential dosage of \-Ki-ras in all tumors inthis system, amplification of the c-Ki-ras gene in normal ratfibroblasts correlated with increased tumorigenicity; further

more, this mutant c-Ki-ras gene in specific cultured subpopulations at low copy number underwent amplification in resultanttumors (26). Increased levels of Harvey-ros were observed inprimary tumors but analyses of metastatic foci found variationin ra.vexpression (23). Chang et al. (16) found that linkage ofthe normal c-Ha-ros gene to an LTR increased the levels of theproto-oncogene product and tumorigenic transformation ofNIH 3T3 cells. In human cells the amount of mutant ras geneproduct (p21) determined the state of transformation (10).These observations suggest that a minimum level of ras geneproduct is critical for early events in the tumorigenic process(23, 27) and the data described here for the Kirsten-ros/BALB/e 3T3 system support this argument.

Some studies have demonstrated that the metastatic pheno-type can be conferred to nonneoplastic cells by transfectionwith tumor DNA (29), "activated" ras oncogenes (21, 23-25,

27) with exceptions in certain cell types (22), or kinase-encodingoncogenes (28). A basal level of ras expression is needed forestablishment of the metastatic phenotype, since in the majorityof studies no clear increases in ras expression were observed inthe metastatic lesions as compared with the primary lesions(30) or with the originally injected cells (23, 31, 33). In somecases, ras may be acting synergistically with other genes inconferring the metastatic phenotype (31). These data are consistent with a model that ras is required for the induction and/or establishment of the metastatic phenotype in certain tumorclasses but may not be needed for subsequent growth of cells atsecondary sites.

The data described here are in agreement with the abovefindings and suggest additionally that Ki-/-Â«.vrearrangements

correlate with enhanced metastasis (33). However, differentmechanisms relating new proviral insertions to growth at thesecondary site might be invoked. Viral promoter insertion withsubsequent activation of specific host cell genes that regulategrowth and differentiation remains a possibility in this systemfor more aggressive invasion and growth in the lung. Alternatively, a strong retroviral promoter or enhancer may facilitategenotypic instability leading to the formation of new variants(71). More detailed molecular biological and virologica! analyses of the tumor populations described here should resolvethese possibilities.

ACKNOWLEDGMENTS

We would like to acknowledge Dr. D. Setzer of this department forthe suggestion of donali) analyzing the tumor cell populations; Drs.T. Pretlow and J. Carter of the Institute of Pathology, Case WesternReserve University, for histology of tissue sections; Dr. H-J. Kung ofthis department for the v-Ki-ras and LTR-specific probes; Dr. D.George of the Department of Human Genetics, University of Pennsylvania, School of Medicine, for the murine c-Ki-ras-specific probe; Dr.T. W. Nilsen of this department for the jird actin-specific cDNA probe;and Dr. B. Yen-Lieberman of the Department of Immunology andCancer Research, Cleveland Clinic Foundation, for purified MoMuLV.

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1988;48:4941-4953. Cancer Res Robert Radinsky, Paul M. Kraemer, Max R. Proffitt, et al. Amplification and RearrangementProgression in Athymic Nude Mice: Mechanisms of

Oncogene during TumorrasClonal Diversity of the Kirsten-

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