(CANCER RESEARCH 50, 760-765. Februar)' I. 1990]

Karyotypic Changes with Neoplastia Conversion in Morphologically TransformedGolden Hamster Embryo Cells Induced by X-Rays1

Musami Watanabe,2 Keiji Suzuki, and Seiji Kodama

Division of Radiation Biology, School of Medicine, Yokohama City University, 3-9 Fukuura, Kana:awa-ku, Yokohama 236, Japan f\t. H'., K. S./, and Department of

Radiation Biophysics, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki 852, Japan fS. K.J

ABSTRACT

Chromosomes from nine morphologically transformed (Ml) cell lines(designated MT14 to MT22) of Golden hamster embryo cells induced byX-rays and from tumor-derived cell lines (MT14T to MT22T), obtainedafter injection of Ml cells, were analyzed by the Giemsa banding method.Ml cell lines showed a variety of numerical abnormalities. All of theMl cell lines involved trisomy of chromosomes 11 (80 to 100% of cellsin each cell line) and 3 (8% of MT22 cells and 100% in other cell lines).Although the latent period for tumor growth differed greatly, eight ofnine Ml cell lines (Ml 14 to MT21) produced tumors at the site ofinjection. All tumor-derived cell lines involved trisomy of chromosome 3at a 100% rate of incidence. Seven of nine tumor-derived cell lines(MT15T to MT18T, MT20T to MT22T) lost one chromosome 11 fromthe trisomie condition, resulting in disomy of chromosome 11. Theseresults suggest that trisomies of chromosomes 11 and 3 may play a rolein X-ray-induced neoplastic progression.

INTRODUCTION

A malignant cell population usually displays some degree ofkaryotypic instability (1-16). The trisomy of a specific chromosome has often been reported in cells derived from humancancers (1-4), animal cancers (5), and transformed rodent cellsin vitro (6-13). Numerical chromosome changes may affectneoplastic progression by resulting in changes in gene dosage,changes in gene balance, phenotypic expression of recessivemutations, or by causing genetic instability (6-8, 14-17). Wehave reported that X-ray-induced, anchorage-independent cellsisolated from colonies in soft agar involved trisomy of chromosome 7, and they showed an additional chromosomal change(trisomy of chromosome 9) in their malignant progenies afterinjection into nude mice (17). These results suggested thatnumerical chromosome changes may affect neoplastic progression. Anchorage-independent cells, however, appeared onlyafter extensive subculturing (17-20). Therefore, the question ofwhether these chromosomal changes are mainly causal factorsin carcinogenesis remains unanswered.

On the other hand, it is well known that morphologicaltransformation is the first observable change in carcinogen-treated cells and that their progenies transform into malignantcells (6-8, 18-24). Several investigators reported that an increase in the number of chromosome 11 relative to otherchromosomes was important in the tumorigenicity of MT' cells

from Syrian hamster embryo cells induced by chemicals (6-8,10). However, Dipaolo and coworkers (25-29), upon surveyinga large number of chemical-induced MT cell lines, failed todiscern any specific chromosomal changes in this or otherchromosomes. Thus, the role of chromosomal changes in neo-

Received 6/15/89; revised 10/10/89: accepted 10/24/89.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.

1This research was supported in part by a Grant-in-Aid for Cancer Researchfrom the Ministry of Education. Science, and Culture of Japan: by a Grant-in-Aid from the Technology Agency of Japan: and by a grant from the HealthResearch Foundation.

•¿�To whom requests for reprints should be addressed.3The abbreviations used are: MT. morphologically transformed; GHE. Syrian

Golden hamster embrvo.

plastic transformation of chemically transformed (Syrian)Golden hamster embryo cells is still unclear. Furthermore,whether the occurrence of karyotype change is of primary orsecondary importance to any steps, such as the initiation,promotion, and establishment of a tumor, has not been resolved.There is strong evidence to indicate that most cancers progressstep by step (18-20, 30). To determine the exact role of anumerical change in tumorigenesis, the most important approach to the problem is to study chromosomal changes occurring at each step, especially at the early steps of malignanttransformation, or to characterize the karyotypes of highlyevolved tumorigenic populations.

The purpose of this paper is to analyze the chromosomalchanges in X-ray-induced MT cell lines and tumor-derived celllines obtained by injection and to examine the relation betweenkaryotype changes and expression of tumorigenic phenotypesin vitro.

MATERIALS AND METHODS

Cells. Cell culture methods and the transformation assays have beendescribed (21, 22). Nine MT cell lines (MT14 to MT22) were clonedfrom morphologically altered colonies of GHE cells irradiated with X-rays (182 kVp) at doses of 0.1 to 4 Gy (21), and 9 tumor-derived celllines (MT14T to MT22T) obtained after injecting them were used. Allcells were cultured in Eagle's minimal essential medium supplemented

with 10% fetal bovine serum and transferred successively.Tumorigenicity Assays. The ability of each MT cell line to form

tumors was assayed by injecting s.c. 2 x IO6 exponentially growingcells (IO7cells for MT22) into the back skin of three nude mice(BALB/c-nu/nu). The mice were examined for tumors weekly. When tumorsreached 1 cm in diameter, they were returned to culture and referred toas tumor-derived cells (MT14T to MT22T).

Chromosome Analysis. Chromosome preparations were prepared bya standard air-drying method and were banded according to a Giemsabanding method as previously described (17). We scored the number ofchromosomes in at least 100 metaphases per sample. For each cell line,at least 20 karyotypes which had the first modal number of chromosomes were photographed and analyzed as described (26).

DNA Transfection Procedure. The calcium phosphate-mediatedDNA transfection procedure (31) was used with minor modificationsas reported (17). NIH 3T3 cells were plated at 1 x IO6cells per 50-cm2

culture dish. Twenty-four h after plating. 30 tig of genomic DNAprepared as described by Wiegler et al. (31) were resolved in a bufferedsolution (21 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid:137 m\i NaCI:0.7 mM Na2HPO4:12% H2O, pH 7.0). sheared by 5passages through a 22-gauge needle, precipitated with 1/10 volume of2.5 M CaCl2, and added to each dish. After 8 h of incubation, themedium was removed, and the cells were treated with 3 ml of 15%glycerol for 1 min at room temperature. Cells were rinsed and readmin-istered fresh medium. Transfected cells were trypsinized 24 h later andreseeded into three dishes for focus formation.

RESULTS

Chromosome Number and Karyotypes of MT Cell Lines. Weexamined chromosome number and karyotype in 9 differentMT cell lines obtained from morphologically altered colonies

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induced by X-rays. All of the MT cell lines increased theirchromosome number and had near-diploid chromosome modes(Fig. la). The 21 pairs of autosomes and 2 sex chromosomesthat constitute the normal hamster complement were clearlyidentifiable. MT cells showed a variety of numerical abnormalities (Table 1). The distribution of the markers in cells rangedfrom no marker to as many as 4 markers. All MT cell lines hadan extra chromosome 11 in 80 to 100% of the cells. In 8 of the9 MT cell lines, all cells had an extra chromosome 3. In theMT22 cell line, however, only 8% of the cells had an extrachromosome 3. MT15, MT17, MT18, and MT22 cell lines hadone to 3 extra chromosomes 8, 19, and 21 in 80 to 100% of thecells. MT16, MT19, MT20, and MT21 cell lines lost chromosome 15 in 80 to 100% of the cells.

Tumorigenicity of MT Cell Lines. When 2 x 10" MT cells

were injected into nude mice, 8 of 9 MT cell lines grew in nudemice at the site of injection, and the latent period for tumorgrowth up to 10 mm in diameter differed greatly between 25and 72 days (Fig. 2). MT15 and MT19 started to grow rapidlyafter a short lag time (10 to 17 days) for tumor growth.Although MT14, MT16 to MT18, MT20, and MT21 hadalmost the same lag times (9 to 25 days) as those for MT15and MT19, they grew slower than MT15 and MT19 during the

a50505050U

SOI

soZ5050SO50-

1 CHE_

MTlt4MT15l

MT16MT17•

rfrMT18MTI94rMT20MT21MT22

-1

b100500so0

50

0so0so0

50

0

50050

0

50

0

50-

1 CHEMTT4TMT15TJLT•

MT16TJMT17TI

MT18TI

MT19T-

IMT20Ti

MT21TMT22T

44 SO 56 62 68 7<4

Number of chromosomes

«1 50 56 62 68 7« ÕNumber of chromosomes

Fig. 1. Chromosome distributions of morphologically transformed cell lines(MT14 to MT22) of Golden hamster embryo cells induced by X-rays (a) and oftumor-derived cell lines (MTI4T to MT22T) (ft) obtained after injection ofmorphologically transformed cells.

first 30 to 50 days after injection following exponential growth.On the other hand, MT22 did not grow at all for 220 days afterinjection. However, if IO7 cells of MT22 were injected into

nude mice, they grew quickly, and the latent period was 53days.

Chromosome Number and Karyotype in Tumor-derived CellLines. The tumors were returned to culture for examination ofthe chromosome number and the karyotype of tumor-derivedcell lines. All of the tumor-derived cell lines have near-diploidchromosome modes and increased their chromosome numbers(Fig. \b). Karyotype analysis showed that tumor-derived cellsshowed a variety of numerical abnormalities, the same as withMT cells (Table 2). MT14T, MT17T, MT18T, and MT19Tcells exhibited trisomy of chromosome 3. The other cells didnot involve trisomy of intact chromosome 3, but they had achromosome 3q- (Fig. 3). The incidence of cells showing trisomy of chromosomes 19 and monosomy of chromosome 15clearly increased. On the other hand, 7 of 9 tumor-derived celllines (MT16T to MT18T, MT20T to MT22T) lost one chromosome 11 from the trisomie condition in MT cell lines,resulting in disomy of chromosome 11.

Transfection of Genomic DNA from MT Cells. In order todetermine whether some endogenous oncogenes had mutatedin the DNA of MT cell lines, we tested the focus-forming abilityof NIH 3T3 cells transfected with high-molecular-weight DNAextracted from both normal and MT cells. As shown in Table3, the ability to transform NIH 3T3 cells could not be detectedin any genomic DNA tested, whereas the DNA from the T24bladder carcinoma cell line, which served as a positive control,demonstrated efficient transformation.

DISCUSSION

We examined the karyotypic changes in cells derived frommorphologically altered colonies induced by X-rays and tumor-

0 10 20 30 40 50 60 70 80 90 100 110

Days after injection

Fig. 2. Tumor growth in nude mice after injection of morphologically transformed Golden hamster embryo cells. Results presented »erethe mean diameterof tumors grown in 3 independent mice. A, MT14: O, MT15; A, MTI6; •¿�MTI7; A, MT18;«, MT19;C, MT20; D, MT21; C, MT22.

Table 1 Karyotypes and latent time for tumor growth in nude mice of X-ray-induced morphologically transformed golden hamster embryo cells

CellsMT14MT15MT16MT17MT18MT19MT20MT21MT22Consistentchromosomeabnormalities"+3.+3,+3q-.+3.+3,+3,+3,+3,-8,+8,+8,+8,+4.

+7. +8, +9.+

11+11,+

10.+I1.-12.+11.+11,+

11,+10,+11.+10,

+11.+11,-15,

-18-15,-15-15++19.+21+++19,

+21++19,+21++19,

+21+

19, +21Construction

of sexchromosomeXXXXXXXXXYXXXXXXNo.

of markerchromosome1140201l0Modal

chromosomeno.4751465151SO464652Mean

latenttime*7235604555256265>220

°Chromosome abnormalities observed in over 80% of cells. +, cells have one extra chromosome; ++. two extra chromosomes; +++, three extra chromosomes;cells lost one chromosome; q-. deletion of long arm of a chromosome (see Fig. 3).

* Mean latent time (days) for tumor growth to 1 cm in diameter.

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Table 2 Kaiyotypes of tumor-derived cells of morphologically transformed Golden hamster embryo cells

CellsMT14TMT15TMT16TMT17TMT18TMT19TMT20TMT21TMT22TConsistentchromosomeabnormalities"+3,

+9,+ 10,+lI,+3q-.+3q-,

+10,+3.+3,

+8,+3,+11.+3q-.+3q-,-6.+3q-,

+7. +9,+ 10,-15,+

17,-15,+

12.+14q+,+17,-15.-12.

-15,-12,-15,+

19,++19.+

18.++19.++19,+

19,+19-18,++

19,+21+21+

20.+21+21+21+21+20+21Construction

of sexchromosomeXXXXXXXXXYXXXXXXXNo.

of markerchromosome313011332Modal

chromosomeno.475147495049464651

°Chromosome abnormalities observed in over 80% of cells. +, cells have one extra chromosome; ++, two extra chromosomes; -, cells lost a chromosome; q-

deletion of long arm of a chromosome; q+, rearrangement of long arm of a chromosome.

derived cells obtained after their injection. The sole karyotypicchanges in both MT and tumor-derived cells were nonrandomnumerical changes (Figs. 4 and 5). The common chromosomalchanges in both MT and tumor-derived cell lines were trisomiesof chromosomes 3 and 11. These nonrandom changes may playan important role in tumorigenic progression.

It is worth noting that trisomy of chromosome 11 was foundin all MT cell lines to a high extent (80 to 100% of cells in eachcell line). These findings can be compared with those of others

who have analyzed transformed Syrian hamster embryo celllines induced by chemicals (6-9). Sachs and coworkers (6-8)and Benedict (9) reported that an increase in the number ofchromosome 11 relative to other chromosomes was importantin the tumorigenicity of progenies of MT cell lines. Theyspeculated that the tumorigenicity of Syrian hamster cells wascontrolled by a balance between genes for expression andsuppression of malignancy. On the other hand, Barrett andcoworkers (10-13) reported that MT cells induced by chemicals

2 3A

n11 12

M13C

MT20T

7 8B

i14 15

16

it17

lu II

D18 19

10

M1 M2 M3

20 21-E- -F-

Fig. 3. G-banding karyotype of tumor-derived MT20T cells involved an extra chromosome 3q—.

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Table 3 Transforming ability of high-molecular-weight DNAfrom normal andmorphologically transformed Golden hamster embryo cells examined by focus

formation assay using NIH 3T3 cells

DNAdonorNo

DNAT24»CHEMT14MT15MT16MT17MTI8MT19MT20MT21MT22No.

of foci/no. ofplates0/64/30/30/30/30/30/30/30/30/30/30/3No.

of foci//igofDNA°<0.0330.13<0.033<0.033<0.033<0.033<0.033<0.033<0.033<0.033<0.033<0.033

°Thirty >igof DNA extracted from various transformed cells were transfectedinto NIH 3T3 cells by the calcium phosphate-mediated transfection procedure.

* Human bladder carcinoma cell line.

exhibited trisomy of chromosome 11 and were immortal, butwere not tumorigenic, and that when the cells progressed totumorigenicity, the extra copy of this chromosome was notalways evident. Our results presented here are consistent withtheir results. In our results, although all MT cell lines had anextra chromosome 11,7 tumor-derived cells lost a chromosome11, reverting the cells from trisomy of chromosome 11 todisomy of chromosome 11. The neoplastic potential of revertedcells did not change after the loss of one copy of chromosome11 occurred. These results suggest that an additional chromosome 11 was acquired in MT cell lines at an early stage ofcarcinogenesis, but this is not directly responsible for tumorigenicity. Several possibilities for these findings should be considered. It is possible that the cells with an extra chromosome11 were not the progenitors for the tumor cells. This is unlikely,because, in MT15, MT17, and MT18, independent karyotypicchanges, i.e., trisomies of chromosomes 3, 19, and 21, wereobserved in both MT and tumor-derived populations. There isno probability that these same karyotypic alterations occurredin three subpopulations. A more possible explanation has beenproposed concerning double nondisjunction in this event (13,32). Tumor cells arising from MT cells may lose the extrachromosome 11 during tumor formation by a second nondis-junctional event. If there is a critical locus on chromosome 11of Golden hamster cells which is heterozygous, double nondisjunction would facilitate homozygosity of this locus, and thismay yield a selective advantage for the cell in vivo. The acquisition of homozygosity of heterozygous markers on specificchromosomes in human tumors by a double nondisjunctionmechanism has been shown for several cancers (33).

Next, we direct our attention to chromosome 3. All tumor-derived cell lines had an extra chromosome 3 or a chromosome3q—at a 100% rate of incidence. Eight of the 9 MT cell lines

showed trisomy of chromosome 3 with an incidence of 100%.Although the latent period for tumor growth differed greatly,when 2 x IO6 MT cells were injected into nude mice, tumors

were produced at the site of injection. MT22 cells, however,had an extra chromosome 3 in only 8% of the cells and did notgrow in nude mice when 2 x IO6 cells were injected (Fig. 2).However, when IO7 MT22 cells were injected into nude mice,

tumors grew in nude mice, and tumor-derived MT22T cellsgained a chromosome 3q-. These findings suggest that trisomyof chromosome 3 rather than trisomy of chromosome 11 isrelevant to tumorigenicity in X-ray-induced transformants.Spontaneous transformation of GHE cells in culture occursonly rarely, and cells cloned from normal colonies becomesenescent at around 30 to 35 cell population doubling levels

(21). Because trisomy of chromosome 11 occurred before senescence of unirradiated GHE cells (at about 25 cell populationdoubling levels), this abnormality may be of importance withrespect to the acquisition of cell immortality.

In most of the cell lines, additional chromosome changeswere found. Cells which had trisomies of chromosomes 3, 19,and 21 (MT15, MT17, MT18, and MT19) could grow fasterthan the other cells. MT19 which had the highest growth abilityin vivo lost one chromosome 15. Cells which showed thesechromosomal changes increased after extensive cell doubling invivo. Thus, chromosomes 19 and 21 may play a role as "ex-pressor" and 15 as "suppressor." Because these additional

chromosomal changes are not essential to express tumorigenicity, they may be responsible for the progression of malignanttumors. Our results were consistent with results which reported(6-9) that the tumorigenicity of Syrian hamster cells was controlled by a balance between genes for expression and suppression of malignancy. Sachs and coworkers (6-8) speculated thatan expressor gene was located on chromosome 11, and asuppressor gene was on chromosome 16. Furthermore, Benedict et al. (34) suggested that the putative suppressor gene in 1-0-D-arabinofuranosylcytosine-transformed cell lines was assigned to chromosome 17. Oshimura and coworkers (11) reported that transformed GHE cells by v-Ha-ras and \-myconcogenes lost one chromosome 15 to convert to tumorigenicityand speculated that chromosome 15 is a suppressor chromosome. Ozawa et al. (13) reported frequent gains of chromosome19 in tumorigenic Syrian hamster cell lines induced by dieth-ylstilbestrol. We previously reported that all transformed GHEcells isolated from soft agar medium exhibited trisomy ofchromosome 7 and that their tumorigenic counterparts wereaccompanied by trisomy of chromosome 9 at an incidence of100% (17). Therefore, we speculated that trisomy of chromosome 7 is relevant to the ability of anchorage-independentgrowth in X-irradiated GHE cells and that tumorigenicity couldbe associated with trisomy of chromosome 9. However, the

Number of chromosomes

Fig. 4. Karyotype of morphologically transformed cells (MTI4 to MT22). Alldata were taken from Table 1. IÀ,monosomy; D, disomy; •¿�-trisomy; (B, 3q—);fJ, tetrasomy; B. pentasomy; E. none.

Fig. 5. Karyotype of tumor-derived cells (MT14T to MT22T). All data weretaken from Table 2. A. monosomy; D, disomy; •¿�trisomy; (B, 3q-); rj, tetrasomy;Q. none.

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progenies of all MT cells tested in the present study, whichwere not accompanied by both trisomies of chromosomes 7 and9, produced colonies in soft agar medium. These findings, takentogether with data presented by others (1-9,11,13,34), supportthe hypothesis that there are many various sequential ordersfor acquiring tumorigenic phenotypes and many combinationsof chromosomal abnormalities expressing each tumorigenicphenotype. Furthermore, our results indicate that additionalnumerical changes are needed for extensive malignant conversion.

Numerical changes of chromosomes as reported in this paperhave been observed in a variety of both animal (5, 35) andhuman (1-4, 36, 37) tumors. However, the question of whetheror not these chromosomal abnormalities are causal factors incarcinogenesis remains unanswered. A possible role for trisomyor monosomy formation is that it may predispose the cell tomitotic nondisjunction, resulting in cells which are homozygousfor specific genes on changed chromosomes (32). Wirschubskyet al. (38) reported that the progression of tumorigenicity wasclosely related to the relative balance between chromosomesthat carry a mutated oncogenic gene locus (c-myc) and itsnormal counterpart which has a decisive influence on theexpression of the tumorigenic phenotypes in a spontaneousAKR-derived thymic lymphoma. The remarkable stability ofthe normal human karyotype suggests that any chromosomalinstability represents a significant departure from the normalcellular phenotype (36, 37, 39). A change in chromosomenumber resulting in activation of an endogenous oncogene mayeffect progression toward a tumorigenic phenotype. In fact,many investigators reported that the activation of oncogeneswas required for immortalization and development of malignancy (40-43). However, no direct evidence that numericalchanges of chromosomes are accompanied by amplificationand/or activation of endogenous oncogenes has been reported.We could not find any mutations of endogenous oncogenes inany of the MT cells related to expression of transformingphenotypes by DNA-transfecting assay using NIH 3T3 cells(Table 3) or any amplification and/or activation of \-myc- andv-Ha-ras-related genes by RNA dot blotting methods (data notshown).

In conclusion, our results emphasize that tumorigenicity mayresult from a balance of expressor and suppressor chromosomesas reported (6-9, 32, 34, 35, 44). Trisomy of chromosome 11resulting from X-ray-induced aneuploidy may play an important role in immortality and trisomy of chromosome 3 intumorigenic conversion. Addditional numerical changes (trisomies 19 and 21, and monosomy 15) are needed for extensivemalignant conversion. The in vitro transformation system offersan opportunity for further analysis of important questionsregarding the role of specific chromosomal abnormalities anddefects in differentiation, in relation to tumor progression andin vitro immortality.

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1990;50:760-765. Cancer Res   Masami Watanabe, Keiji Suzuki and Seiji Kodama  Induced by X-RaysMorphologically Transformed Golden Hamster Embryo Cells Karyotypic Changes with Neoplastic Conversion in

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