Karyotype evolution in a simian virus 40-transformed tumorigenic human cell line

Karyotype Evolution in a Simian Virus 40-Transformed Tumorigenic Human Cell Line Charles L. Goolsby, John E. Wiley, Marianne Steiner, Marry F. Bartholdi, L. Scott Cram, and Paul M. Kraemer

A B S T R A C T : Normal h m n ( m lorcskin t]l)rol)l(~sts (t1S1:4) w(u'e trm~h,(:ted t]si))!~ the f)SV3-m!() ))l()~mi(l. A I)o()l ~)t 1(1 (;41~-rcsisi(mt (:()l(mi~!s. tl,'c,l"4-T12, sh()w(!d (~ pro!Lr(~ssh'(> iH(:rt!(lm! it~ the (~xl)r(~ssi(m ol (~ m.)ml)er of in vitro [l'(lllSI})l'Ill(IIit.)l) /))(irk(~)'s with pt).'~s(~(~ h~ ctdtm'c (rod b(~(:mnc itl)ll)ortaliz~(l. A l t l l o t l ~ h n(.) [tltl)ol's w(~t'(~ I(;l'l]')(~(/ t,.'l)t~tl ceils r,'cre il)ict:lt'd sul)(:tlltzt~ct)t)~]~, it)It) mr(to mi(:c, this (:ell lh~c tm)(lt)(:c(l t)ro,~4r(~ssivc tmnors whet) (:ells were h)i('(:tcd h)l~) l)reiml)hmted (h!Ifo(-m) ~l)()nges it) lh(! mi(:(!. Wh(m lh~!.~c ttm)t)rs were (:ulh~r(~(l i)) vitr() (md szll),'~(!qtletdly illj(,'(;led .~lll)t:tlt(lll(~Otl,'41~'. t)ro(,~'r(!,~siv~!h~zl)or~ tvt?)'(~l)rt)(lllt3~(] with mt~(tim) hmm(:v

(~a)'ly l)assag(~s (dler t)'(msfc(:tio)). I I,".;l,'.;-'l'l2 exhibited )ncmx" r(m(l(m~ (:hromoso.)cd ( h(m,_,es. At (( time just a.lter immortaliz(dio)). /toll)Ihtw k(~rvoh'l m m)d G-b(m(h~d m)(dvscs showed the (~t)p(!m'(mcc of t)(dcmc(!(t (:hm(ll rc(m'an,~,emc))ts. Thes(! im:h)(te(l t12;4L II2.:14}, tFl:i'L (~1~- . i((il)). ,~lI) . 1(14;15). i(151, and tl l,~):!'). "l'h(~s~ (:lomd rc(.'r(m!,~(!mct)ts were ~t()l~](! with l~(t.~s(tS~'~) ix) (:tdlure. (tt)(l le.'~s vari(d)ility Irt)m (:(~II to (:vll ix(is ~)()tc(l. The ( m h (:()m;isR~))( (:hr(tm(tmm)(d loss t~l)s(~rx'(~(l w(xs 'f. A)~ah'sis ()I' thrc(~ i))del)(~mh~nt ttm)()rs ~h()w('d (:h(xt'(.:t(,risti(: l(~s~ (d chrt)llloSoll)(i] l)l(It(~l'i(l] r{ltht!r th(ll! ])(I](lllc(!(t (:l)l'(tll)O,~()lll(l] r(!(llT(lll/-(~lllt!tl[~, l"l'l!(tu('Id l()s~ ol (~(l (ll)d (hron)oso)m~s # I : h 15. 20. (md Y wtl,~ ))ote(l.

INTRODUCTION Current (:ytogenetic evi(len(:e implies that (:hromosoma[ rearral~gements [)lay aa i m p o r t a n t role in t h e h u m a n neol)lasti(: l)ro(:ess. Numer(m.'-; spe(:ifi('. (:hr()m()somal (:hal~.ges h a v e b e e n a s s o c i a t e d w i t h h u m a n lm~ligt~all.(:i(-;s, i n ( : l u d i n ~ soli(t h ] m t ) r s , l y m p h o m a s , a n d l e u k e m i a s [1, 2]. T h e s e sl)e(:ifi(: ( : h r o m o s o m a l c h a n g e s are freq u e n t l y o b s e r v e d in c o n i u n ( : t i ( ) n w i t h n o n s p e c i f i ( : c h r o m o s o m a l r e a r r a n g e m e n t s . refle(:ling o n g o i n g k a r y o t y p e i n s t a l ) i l i t v , in s ) u ( l i e s of ~(:~)te m y ( ~ h ) g e n o u s h : u k e m i ~ (AML). a(:ute l y m p h o b l a s t i ( : l e u k e m i a (ALL), an(t n o n - H o ( I g k i n ' s l y m l ) h o m a , 70 9 0 % of t h e c a s e s (;xhibite(l c h r o m o s o m a l a b n ( ) r m a l i / i e s [3 7: r(;view(t(t in t e l

Irr()m the l)(~l)artm(,)t o| l)i)tl.)lo!~'v. N(wlhw(~t.r. [tni~ (~]silv .x.1(~di(:al ,"4(ho()l 'VA l,.k.~i(h~ N,1(~(lic:~)l( ;(.]r(w ((LI,.(;., M ,'#,.),(]hi~:ag(). lllin()i>-;,the D(~Farl[mmt ol l).diatri(:>,'(;(..qi(:s, l'la~.t (]arolm. ttni~.(w>.ih' S<:h()()1 ()l M(~(li(:i))(~ () E.Vv"). (;r(~(~)wilh~. Nodh {k)r()lina. ~md (~(>II Bh)h)~v (.r()~q). I.os ALm~()s N~|i~m.l t.a~(.ah))-'. [M.I:.B., I,.S.C.. I).XI K.). l,os Ah[m()s. N~.,v M(~xi(().

Ad(h'ess t't~l)rittt t'(~tlt1(tsts tt~: l)r. (3)arl(~s Gt)tdsltv. l~(.)t)) 711, V A L((ktrsi(hr Mcdi(:(d Cc))((w. ?)3:1 I",, lh)ron St., (]hh:()go. IL (;()(~l I. l~(~('eivt~d l;(~brmlrv 1, 19!,)0: (J(:(:t~l)tt~(l A.l)ril 19, 1!1!t0.

231 (kh5 /',.v(!nm! o[ l h e Am(wi(:a~,. N%w Y o r k . N Y ](10 ] 0

() I(;5 -l(i()~,91 S 0 3 5 0

232

(:. l,. ( ; o o l s b v et al.

21, and de la Chapelle [2] has suggested that the remaining cases would probably show m o l e c u l a r rearrangements. Owing primarily to difficulties with the in vitro growth of the cells, h u m a n solid tumors have been much less well studied than leukemias and l y m p h o m a s . Data on karyotypic changes in h u m a n solid tumors are hard to interpret in terms of the role these changes may play in the neoplastic process owing both to the large number of changes that occur and to the generally late stage ill the neoplastic process when (:ells are examine(t. Specific (:hron/oson~al changes have been associate(t with some solid tumors however [1, 2. 8 101. In addition, studies using cells derived from human tumors yiehl insufficient informalion to clarify whether these chromosomal changes precede or accompany initiation or are, secondary changes selected during later steps of the neoplastic process. Monitoring karyotypic changes during transformation of primary normal h u m a n fibroblasts or epithelial cells in vitro may provide some insight into the role that chromosomal changes may play in the acquisition of various transformed phenotypes. Studies of this nature have been difficult with h u m a n cells owing both to difficulties with the immortalization and the tumorigenic steps in transformation. Although n u m e r o u s studies have investigated karyotypic changes in transformed h u m a n cell lines, seldom have these studies been able to monitor the changes in a systematic way from the normal p h e n o t y p e through the various stages of transformation, immortalization, and tumorigenicity within one cell line. Simian virus 40 (SV40) transformation of both h u m a n fibroblasts and epithelial cells has been studied for more than 25 years. Infection with SV40 virus or transfection with the early genes of SV40 are known to induce numerous karyotypic changes [11]. A l t h o u g h some reports have described specific chromosomal changes associated with these processes [12-14] and recent studies have reported some c o m m o n patterns in the types of rearrangements observed in SV40-transformed h u m a n cells [15-17], most studies have c o n c l u d e d that the chromosomal changes differ from lineage to lineage [11, 18-20]. Many of these studies have used an in vitro measure of transformation such as colony formation in soft agar, foci formation, or i m m o r t a l i z a t i o n to screen cells and thus have not been able to examine early karyotypic events following the expression of T antigen. In addition, acquisition of the tumorigenic p h e n o t y p e has been rare after SV40 transformation of h u m a n cells [21]. In a recent study by Wu et al. [17], the tumorigenic p h e n o t y p e was i n d u c e d in a SV40-immortalized h u m a n uroepithelial cell line (SV-HUC-1) by treatment with the chemical methylcholanthrene. The growth of tumors, formed by subcutaneous injection of these cells into nude mice, was a c c o m p a n i e d by a n u m b e r of u n b a l a n c e d chromosomal rearrangements. In contrast, in vitro culturing of the SV-HUC-1 cell line was a c c o m p a n i e d by a number of generally balanced c h r o m o s o m a l rearrangements [16]. In the present study, a h u m a n fibroblast cell line, HSF4-T12, was established by transfection with the plasmid, pSV3-neo, w h i c h contains the early genes of SV40 as well as the n e o m y c i n resistance gene. Because selection of successfully transfected cells was based on growth in G418 containing m e d i a rather than immortalization or an in vitro transformation measurement, we were able to examine karyotypic changes that occurred in the T - a n t i g e n - p o s i t i v e cells before i m m o r t a l i z a t i o n or significant expression of a transformed phenotype. In addition, HSF4-T12 after 136 p o p u l a t i o n doublings (PDs) p r o d u c e d progressive tumors w h e n cells were injected into p r e i m p l a n t e d Gelfoam sponges in n u d e mice. Karyotypic changes in four i n d e p e n d e n t tumors of HSF4-T12 were also examined. Thus, our analysis includes the karyotypic changes before immortalization or significant transformation, just after immortalization, with long-term in vitro culture, and in tumor cells derived from the same cell line. The results suggest that three distinct phases of cytogenetic change were involved in the mnltistep neoplastic transformation of h u m a n cells transfected with the early genes of SV40.

Karyotype of a Tumorigenic H u m a n Cell Line

233

MATERIALS AND METHODS Cells and Culture Methods The normal HSF4 strain used in these experiments was provided by Dr. David Chen, Genetics Group, Los Alamos National Laboratory, Los Alamos, New Mexico. Cultures of normal cells were grown in ~ - m i n i m u i n essential me(tium ((~-MEM, GIBCO. (;ran(t Island, NY) plus 10% fetal (:alf serum (GIBCO; or Hyclone, Logan, UT]. Cultures of transfecte(I (Jells were (:arried in the same media with the a(htition of 250 /ag/ml active gentamycin ((;418, GIBCO). The cultures of transfected (:ells were routinely subcultured after remaining at confluence for 4 - 7 days at ea(:h passage t(/ give a selective growth advantage to the transformed cells. Cultures were seeded at 1.5 × 10" (Jells per T150 flask (Corning (;lass Works, Coming, NY). After immortalization, the cell line HSF4-T12 was sub(:ulture(t on(:e a week. Cultures were seeded at 7.5 × 10 r' cells per T75 flask (Coming). We t)erformed cell (:ounts on (:ell (:ultures at the time (if subculturing and kept detailed PD records.

Transfections Tbe pSV2-neo and pSV3-neo plasmids were obtained from American Tyl/e Tissue Culture (ATTC, Rockville, MD} collection in Escherichia coil strain HBI(I1. Plasmid DNA was isolated by the method of Birnboim and Doly [22]. HSF4 cell (:ultures were transfected by the method of Maclnnes et al. [231 with minor alterations. Cah:ium phosphate precipitates of plasmid DNA were prepared and 20/,g plasmid DNA were added to 60-ram tissue culture dishes containing 10 ~ HSF4 (:ells. After a 12-hour incubation, the cultures were shocked with media containing 15% glycerol for 3 minutes, washed twice with media, and placed on ~,-MEM plus 1(i% fetal calf serum for 3 days. Cells were then trypsinized and plated at 9 x 105/1Oil-ram tissue culture dish [Falcon, Lin(:ohl, NJ) in selective media. At 21 days, 26 i n d i v i d n a l resistant colonies were ringed, trypsinized, and expanded in T25 flasks {Corning). The (:ell strains established were designated as HSF4-T#, where # equals the nuinber representing the order in which the colonies were isolated. HSF4-T11 and HSF4-T12 were pools of six and 10 resistant colonies, respectively. Alternatively, approximately 3 × 10 ~ HSF4 cells per T75 tissue culture flask were transfected witb 20/,g plasmid DNA as des(:ribed above. After the 3-day expression period on ¢,-MEM ph(s 1(l% fetal calf serum, the cultures were changed to selective media. These cultures were subculture(I when resistant cells had grown to more than 70% confluence.

DNA Content Measurements DNA content nmasurements were made on (Jells fixed in 80% ethanol and stained with 50 p~g/ml propidium iodide in phosphate-buffered saline. Cells were then analyzed on the Coulter Electronics EPICS C flow cytometer of the VA Lakeside Medical Center, Northwestern University Medical School.

Karyotype Analysis Cultures for flow karyotype analysis were blocked with 0.1 /~g/ml Coh:emid for 14 hours. Mitotic cells were then concentrated by mitotic shake off and 5 7 x 10" (Jells were then swelled in 40 mM hypotonic KCI at room temperature for 10 minutes. Chromosomes were then isolated by addition of an equal volume of a twice cou(:entrated buffer containing spermine and spermidine [24J by vortexing. The suspension of chromosomes was then stained with 110 p,M c h r o m a m y c i n A3 for 12-18 hours

234

(:. L. Goolsby et al.

and just before analysis with 2 /~M Hoechst 33258, The sust)ensions were then analyzed on the EPICS 753 flow cytometer at the Northwestern University Medical S(:hool equipped with kryt)ton (Coherent Radiation model 90-K, Palo Alto, CA) anti argon ion (Coherent Ra(tiation model 90-5, Pal() Alto, CA) lasers. Spreads for (:hromosoine counts and G-ban(te(I analyses were prepare(t by traditioiml techniqtms. (;banded analysis was by the trypsin digestion metlmd of Seabrigbt with slight modifi(:ations 1251.

In Vitro Transformation Assays Cultures to be assayed for growth in h)w (:on(:entrations of serum were trypsinize(I and plated in replicate in 1% serum at 2 × 11)4 (:ells/60-mm tissue culture dish, allowed to attach for 24 hours, and then placed on media with various serum concentrations. Replit:ate dishes were the,n (:ounted at the same time on 6 conse(:utive days. A linear least-squares fit was then performed on the exponential growth portion of this data and the PD time (PDT) was cah:ulate(t from the slope, of this line. Growth in any given (:on(:entration of serum was then re(:or(le(t as the ratio of the PDT in that (;oncentrati(m divided bv the PDT in 10% serum. (Mh)nv formation in 1 and 10% serum was determine(1 in a similar manner by see(ling lOl)-mm tissue (:ulture dishes with either 250, 500, i() ~, or It) ~ (:ells. After 21 days, dishes were stained with a solution of 1% crystal violet in 71)'!.i,(,(hanoi, and (:olonies with lnore than 50 (:ells were (:ounted. Saturation density was determine(t [)y allowing (:ultures to rea(:h (:onfluent:e in (~-MEM plus 10% fetal (:all serum, refeeding the (:ultures, and allowing them to grow for an attditional 5 (lays. The (:ultures were then tryl~sinize,(I. (:()unte(I, an(I the n u m b e r of (:ells per square, (:cut(meter was (tetermine(t. Colo.y formation in m e t h v l c e l l u l o s e w a s d e t e r m i n e d b v s u s p e n ( t i n g 3 × lO:' (:ells in IOmI(~-MEM plus 10% fetal calf serum (:ontaining 0.15 g methyh:elluh)se. This suspension was then equally divided into three gridded (i0-11nll tissue (:t(lture dislms (Corning 25011, Corning) (:oated with 0.5% agarose (:ontaining (~-MEM I)lus 10% fetal (:alf serum. After 21 (lays, colonies greater than 1011 /.Llll ill diameter were (:(mnte(t.

Tumorigenicity Tumorigelficity was teste(l by injecting a slurry of (:ells (:ontaining at least I × 107 (:ells into Gelfoam sponges implanted 10 (lays earlier in nude mi(:e 1261 or by stJbcutaneous inje(:tion. Injection of (:ells into imt)lanted sponges provides a significantlv in(:reased sensitivity, parti(:uh~rly in the case of marginally tumorigeni(: huinan (;ells, by providing a wls(;ularized substrate It) which (:ells can attach and progress in vivo. Cells that do not form tumors when injected sub(:(mmeously may form tumors when injet:ted int()t)reiml)hmte(t st)onges [27].

Staining for Large T Antigen The expression of large T antigen was assayed bv standard indirect immunoihlores(:ent techniques 1281. Cells were grown on (:overslips and then fixed by a(:etone. Primary antisera was ascites fluid from a hamster bearing an SV40-indut:ed tmnor (provided bv Dr. ]. But(l. Baylor College of Medicine} or the monoclonal antibody, PAb419 (Oncogene Scient:e, Manhasset, NYI. Fluoresce(n-conjugated goat anti-hamster lgG (Cooper Biome, dical, Cochranville, PA) or fluorescein-(:onjugated goat antimouse IgG [Coulter Immunology, Hialeah, FL) was used as se(:ondary antibody. Negative controls used included HSF4 (:ells, no primary antisera, and a mouse isotype control. The cell line SVT2 (ATTC, Rocky(lie, MD) was used as a positive control. Positive (:ells were then scored micros(:opically.

Karyotype of a T u m o r i g e n i c Human Cell Line

235

The quantitative flow cytometric m e a s u r em en t of T-antigen expression was performed by the method of Jacobberger et al. [291 with slight modifications. Cells were fixed with ice-cold ethanol to a final concentration of 80%. Primary antisera was the monoclonal antibody, PAb 419; the secondary antibody was a fluoresceiu-(:onjugated goat anti-mouse antibody. All negative and positive controls were the same as above. Cells were counterstained with 50 /zg/ml p r o p i d i u m iodide and analyzed on the Coulter Electroni(:s EPICS C at the VA Lakeside Medi(:al Center, Northwestern Universitv Medical School.

RESULTS Expansion of Resistant Colonies (;418 resistant colonies were obtaine(t at a frequency of 1 - 2 / 1 0 ~ cells plated after transfection with either the pSV3-neo (/r the pSV2-ueo (which lacks tile SV40 early genes) plasmids. For the pSV3-ueo transfections, 26 individual resistant (:olonies were ringed, tryl)sinized, and expanded in T25 flasks. All but two of these 26 (:oh)hies wer~~. successfully expanded in selective media to form (:ell strains. All 24 of these strains entered a crisis phase at PD levels between 40 and 67, and none survived crisis to forln lines. Two cell strai,ls were estal)lished by pooling all of the resistant colonies ill a transfected mass culture. In one case, HSF4-T11, six coh)nies were peele(t: in the other, HSF4-T12, 10 colonies were pooled. HSF4-T11 entered crisis at 74 PDs and did not survive. HSF4-T12 entere(I a short and not very obvious crisis t)eriod at approximately 8 5 - 9 0 PDs and now appears tn be a 1)ermanent (:ell line. It has been (:arried for more than 380 PDs and conlinues to gr()w well. The expansion I)eri()(t f()r all (:ell strains was characterized [/y the shedding of many dead (:ells. For tile trausfection with pSV2-neo, 10 individual resistant colonies were ringed, trypsiuized, and expanded in T25 flasks. Seven of these colonies were successfully expanded in selective media to form (:ell strains. In addition, two transfected mass cultures (af)l)roxinmtely six to 10 resistant colonies per culture) we,re als() ext)ande(t in selective media. All these (:ell strains entered a terminal crisis phase at between 40 and 60 PDs.

In Vitro-Transformed Phenotypes Tile expression of the in vitro-transformed t)henotypes ,)f growth in 1 an(l 0.1% serum. colony formation in 1% sermn, increased saturation density (10% sertnn), and colony formation in inethylcellulose were assayed. None of the pgV2-neo-transfected (:ell strains showed any significant changes from the parental strain HSF4 in the expression of these t)arameters. Fifteen of the 26 (:ell strains generated by transfecti(m with pSV3nee were tested for T-antigen expression as soon after transfection as possible (passage 2). All these celt strains were T-antigen positive as ev i d en ced by nuclear fluorescen(:e when stained with either ascites fluid from a SV4t) tumor-bearing lmmster or wilh the monoclonal antibody PAl) 419. The results of the in vitro transformation assays for the (:ell line HSF4-T12 at a number of PD levels and for the parental strain HSF4 are shown in Table 1. Although 100% of the (:ells were T-antigen positive, tile onh, significant increase in any of these parameters for HSF4-T12 at early passages after transfection was an increase in the growth rate in reduced serum cnncentrations. Similar results (data not shown) were observed for four of the indet)endent I)SV3n e o - t r a n s f e c t e d cell strains. The IISF4-T12 lineage showed a progressive in(:rease in the expression of all in vitro-transformed phenotvpes with passage in culture,. The senescen(:e/inmlorhdiza-

236

c . L . (;oolsby et al.

Table 1

Results of in vitro transformation assays for cell line HSF4-T12 and parental line HSF4 (;ro'o.:th I'

Cell strain/line

Pl}s

PIYI'"{h}

1%

CF'

{}.1%

1{}%

1%

Saturation {lensity ~/

HSF4

3{}

26

0.22

0.10

47.{}

HSF4-T12

66

45

{}.47

{}.22

{}.9

HSF4-T12

99

:]8

{}.64

{}.3B

HSF4-T12

115

ND

NI}

NI}

6.{}

HSF4-T12

136

36

{}.{}7

0.51

13.{l

{}.1

,{}.7 x

1{} }

NI}

HSF4-T12

165

35

{}.89

{}.55

22.{}

{).3

2.1

×

l{}:'

{}.6

HSF4-TI2

354

22

0.81

{}.53

21.8

10.7

3.1

×

1{}-'

5.6

ND

<0.01

5.B ×

l(} *

<{}.{}1

6.4

1{} ~

M{~thyl{:el hd{}s{,~{%}''

ND

x NI)

{}.{}8

"3 3

NI}

x

1{}

×

1{}

×

l{}

{}.(}1

A b b r e v i a t i o n s : I ISF4, h u i n a n f o r e s k i n fibroldasts: PDs. i}olmlati{m doublillgS; PI)T, PI) time: CI", ted(my f o r m a t i o n : NI), n{}l {I{}n{h " PIYI' in 10% fetal b o v i n e S{}FIIFI]. J' PlYI' in 1% {0.1%} fetal b o v h ] e serum/PlY}' in l{}'", h}hd I}{}vine s e r u m . ' l}er{:entag{} {}f CF in 1{}% {}r 1% fehd I}ovin{~ s e r u m {}n plasti{: {{:olonies of m o r e t h a n 5l} {:~lls].

" Cells pep square {:{mtimeh}rat {:onfhwn{:e. " P{}r{x!nta~e {}t CF in mt~thyl{:elhdose { 1{}% fetal b{}vh]e Sel'Un]; {:{}h}nies greater thai] 1{}{}~II/).

tion point in this lineage was between approximately 85 90 PDs. lust after ilnmortalization (PD - 99-115), the emergent cell line exhibited significant growtb in reduced serum concentrations and formed slnall but positive colonies in 1% seruin on plastic and in m e t h y l c e l h d o s e containing 10% serum. With continued passage in culture, there was a progressive increase in the expression of all of the in vitro transformation markers assayed. These data show that for pSV3-ueo transfected h u m a n fibroblasts extensive passage in culture may be required for expressiou of most in vitro transformation markers. The increase in the expression of these markers with the in vitro passage of HSF4-T12 was not ac{:ompanied by any significant changes in T-antigen expression as measured by quantitative flow cytometric determinations of immunofluorescent intensity [data not shown).

Tumorigenicity Experiments using both conventional and preinlplanted sponge assays for HSF4-T12 at several PD levels and for two i n d e p e n d e n t pSV3-neo-transfected cell strains that did not become immortalized, HSF4-T4 and HSF4-T9, are shown in Table 2. Both HSF4-T9 and HSF4-TI2 (PD - 63} were negative even when (:ells were injected into preimplanted Gelfoam sponges. In one of the five animals injected with HSF4-T4 (:ells, a small tuinor (approximately 5 mm) developed after 3 months. In the next 2 months, however, the tumor did not increase in size. Just after immortalization, HSF4T12 (PD = 136) produced progressive tumors in eight of 10 animals with a median latency period of 12 weeks w h e n (:ells were injected into preimplanted sponges. All these tumors have been successfully recultured. When HSF4-T12 at a later passage level (PD = 161) was retested by the sponge assay, no increase in the tumorigenicity phenotype was evident. Cells from three of the recultured tnmors (HSF4-T12 T{ J1, HSF4-T12 TU2, and HSF4-T12 TU3) were injected subcutaneously into nude mice. These data are also shown in Table 2. All three cell lines produced progressive tumors with median latency periods as short as 4 weeks. Subcutaneous injection of HSF4T12 (PD - 287) (:ells into nude mice produced no tumors.These data show that HSF4T12 u n d erwe n t a significant progression in the in vivo tumorigenicity phenotype that

237

K a r y o t y p e of a T u m o r i g e n i c H u m a n Cell Line

Table 2

Experiments with conventional and p r e i m p l a n t e d s p o n g e a s s a y s for H S F 4 - T 1 2 at s e v e r a l PD levels, for t h r e e i n d e p e n d e n t t u m o r d e r i v e d cell lines, H S F 4 - T 1 2 TU1, H S F 4 - T 1 2 TU2, a n d HSF4-T12 TU3, a n d for t w o i n d e p e n d e n t p S V 3 - n e o - t r a n s f e c t e d cell s t r a i n s HSF4-T4 a n d H S F 4 - T 9

Cell strain/line,

PDs

HSF4-T4 HSF4-T9 HSF4-T12 HSF4-T12 HSF4-T12 HSF4-TI2 HSF4-T12 TU1 HSF4-T12 TU2 HSF4-T12 TU3

Sponge assay (wk)

51 49 63 136 161 287

Sul3cutaneous assay (wk)

1/5. 14 0/5, ' 3 2 0/5, :-32 8/10, 12 13/2(I, 12 ND NIl NI) ND

ND ND ND NI) ND 0/5, ---32 4/10, 16 9/lO, 4 7/10, 8

Abbreviations as in Table 1. d i d not o c c u r in vitro e v e n w i t h e x t e n s i v e p a s s a g e of HSF4-T12 in c u l t u r e . R e s u l t s of t h e in vitro t r a n s f o r m a t i o n a s s a y s o n four of the cell l i n e s e s t a b l i s h e d f r o m r e c u l t u r e d t u m o r s are s h o w n in T a b l e 3. T h e m o s t s i g n i f i c a n t c h a n g e in all t u m o r - d e r i v e d cell l i n e s w a s i n c r e a s e s in c o l o n y f o r m a t i o n in 1% s e r u m on plastic. T h i s i n c r e a s e for the t u m o r - d e r i v e d c e l l s also o c c u r r e d for H S F 4 - T 1 2 w i t h f u r t h e r p a s s a g e in c u l t u r e . O n e of the t u m o r - d e r i v e d lines, HSF4-T12 TU4, s h o w e d s i g n i f i c a n t r e d u c t i o n s in b o t h s a t u r a t i o n d e n s i t y a n d c o l o n y f o r m a t i o n in m e t h y l c e l l u l o s e .

D N A Content Measurements DNA c o n t e n t w a s m e a s u r e d by flow c y t o n l e t r y for c e l l s f r o m HSF4, HSF4-T12 (PD 63, 1 3 6 , 2 8 0 ) , H S F 4 - T 1 2 TU2, H S F 4 - T 1 2 TU3, HSF4-T12 TU4, a n d HSF4-T12 TU8. D i s t r i b u t i o n s of t h e n u m b e r of cells v e r s u s r e l a t i v e f l u o r e s c e n c e i n t e n s i t y (DNA

Table 3

R e s u l t s of in vitro t r a n s f o r m a t i o n a s s a y s o n four cell l i n e s e s t a b l i s h e d f r o m recultured tumors Growth t'

(;ell strain/line HSF4-T12 HSF4-T12 HSF4-T12 HSF4-T12 HSF4-T12

CF':

PDT ~ (h)

1%

0.1%

10%

1%

Saturation density ~j

35 37 41 44 32

0.89 0.68 0.82 0.88 0.90

I).55 0.58 0.45 0.52 0.72

22.0 9.5 11.3 12.2 9.5

0.3 3.5 2.8 1.5 7.6

2.1 2.0 1.5 5.4 1.5

(PD = 165) TU2 TU3 TU4 TU8

× × × × x

105 105 10" 104 10:'

Abbreviations as in Table l. o PDT in 10% fetal bovine serum. ~'PDT in 1% (0.1%] fetal b o v i n e serum/PDT in 10% fetal bovine serum. Percentage of CF in 10 or 1% fetal bovine serum oil plastic (colonies of more than 50 celIs].

':

'~Cells per square centimeter at confluence. Percentage of CF in methylcellulose (10% fetal bovine serum; colonies greater than 100 ~.m].

Methylcellulose (%)" o.B 0.6 0.2 0.07 ND

238

(:. L, G o o l s b y et al.

u) .J ..J

W

¢J 0

W m IE

c

Z lid

Ill n-

D

RELATIVE DNA CONTENT F i g u r e 1 I)NA (:onlenl deterufillalit,ls. I)anel A: HSF4. n (range ot mnnfl)er of (:hr(m~t)s()mes per (:ell) 46. Palm t:~:HSt:4-T12 (I)opulalion doubliIIgs, PI) 631, n 42 46. Pared C: HSI"4TI2 (PI) : 136).n 37 44. I)allel I): HSV4-TI2(I'I) 2~/0).n 59h) more than g(I. Panell(: 1{$1:4-'1'12 T[J2, [I 41-50, 67-78. Panel l": 11,g1"4-'1'12TtI3, n 40 48, 71 7!). I~an(,q (;: HS1"4'1'12 TIJ4, n 4:{ 45, (18 -75. Prowl It: HSV4-TI2 TIJS, n 43 53.

c o n t e n t ) are s h o w n i n F i g u r e ~1. T h e d i s t r i b u t i o n s h o w n for H S F 4 is t y p i c a l of e x p o n e n t i a l l y g r o w i n g h u m a n f i b r o b l a s t s i n c u l t u r e . H S F 4 - T 1 2 (PD = 63) s h o w s s i g n i f i c a n t i n c r e a s e s in t h e p e r c e n t a g e of S p h a s e a n d G 2 / M p h a s e cells, s u g g e s t i n g a h i g h e r p e r c e n t a g e of c y c l i n g cells as c o m p a r e d w i t h HSF4. A n u m b e r of cells also h a v e D N A c o n t e n t s g r e a t e r t h a n G 2 / M , i n d i c a t i n g t h e a n e u p l o i d n a t u r e of s o m e cells w i t h i n t h e p o p u l a t i o n . H S F 4 - T 1 2 {PD - 136) is s i m i l a r to H S F 4 - T 1 2 (PD - 63) in D N A c o n t e n t , a n d s e l e c t i o n of a d i s t i n c t a n e u p l o i d p o p u l a t i o n at t h i s stage e v i d e n t l y h a s n o t o c c u r r e d . W i t h c o n t i n u e d p a s s a g e in c u l t u r e , t h e s e l e c t i o n of a n e a r - t e t r a p l o i d p o p u l a t i o n w i t h loss of n e a r l y all of t h e d i p l o i d p o p u l a t i o n is e v i d e n t in H S F 4 - T 1 2 (PD = 280). D N A c o n t e n t m e a s u r e m e n t s o n t h e t u m o r - d e r i v e d cell l i n e s d i d n o t s h o w a n y c o n s i s t e n t s e l e c t i o n of e i t h e r t h e d i p l o i d or a n e u p l o i d p o p u l a t i o n s d u r i n g t h e p r o c e s s of t u m o r g r o w t h in n u d e mice. As w i t h H S F 4 - T 1 2 , h o w e v e r , e m e r g e n c e of a n e a r -

Kary{}type of a Tumorigenic Human Cell Line

239

tetraploid p o p u l a t i o n was observed with long-term culturing of these lines (data not shown).

Karyotype Analyses HSF4-T12 Progression In Vitro.

Flow karyotype and G-banded analyses were performed on HSF4 and HSF4-T12 at several passage levels and on cells from tbre.e tunlor-derived cell lines. With parental cells (HSF4}, both flow karyotype [PD 30} and G-t)all(led analyses (PD 25) indicated a normal 46,XY karyatype ((tata m}l shown}. A p o l y m o r p h i c cbrolnosonle 21 was evident in HSF4 an{t HSF4-T12. h(}wever (Figs. 2 and 3).

Early Passage Post-transfection. Figure 2A is a bivariate histogram of Hoechst 33258 fluorescence versus c h r o m a m y c i n A 3 fluorescence for HSF4-T12 at 56 PDs. For HSF4T12, 56 PDs represents a stage before the cells were immortalized, tumorigenic, or significantly transformed, as shown above. At this stage, all the normal c h r o m o s o m e peaks were represented in the bivariate flow karyotype and no new or abnormal peaks were evident. G-banded analyses at this stage showed chromosomal changes in all 22 spreads examined, however. The only consistent chromosomal rearrangement (present in 20 of 22 of the cells by G-banded analysis) was t(3;?)(q27?;?) (Table 4). These data were consistent with the flow karyotype results. For a change to be obvious in the bivariate flow karyotype, it must occur in a significant percentage of the cells [30]. In addition, the rearrangement must be sufficiently large and well defined to produce a noticeable change in the flow karyotype. The t(3;?) observed at this and later stages c o m p r i s e d a subtle and small addition. In addition to the t(3;?), three rearrangements to p r e d o m i n a t e later in the evolution of this line, i(6p), del(6) (p11.2), and t(18;?)(p?;?), were noted in two of 22, three of 22, and four of 22 (respectively) cells. Thus, for rearrangements that can be identified by flow cytometric analysis, bivariate flow cytogenetics provides a method by which the concept of marker c h r o m o s o m e s within a population can be refined and operationally defined.

Early Postimmortalization. At 136 PDs, HSF4-T12 was just postimmortalization, c o n d i t i o n a l l y tumorigenic, and expressed a number of in vitro transformation markers. Four new peaks were obvious in the flow karyotype at this stage, as was loss of the normal c h r o m o s o m e 6 and Y peaks (Fig. 2B). By comparing the size and chromosomal constitution of the consistent rearrangements observed in the G-banded analyses at this stage (Fig. 3A and Table 4), we made probable assignments of the rearrangements to some of the new peaks in the flow karyotype. Two chromosomal rearrangements, t(2p4q) and t(2p14q), were probably contained in the M1 peak observed in the flow karyotype. Both the t(14q15q) and i(15) rearrangements were consistent with the position of the M2 peak. The M3 peak position was consistent with the i(6p). No rearranginent identified in the G-banded analyses was consistent with the M4 peak in the flow karyotype. The del(6)(p11.2) should underlie the X and chromosome 8 regions of the flow karyotype and indeed an increase in the counts and considerable broadening of these areas were noted. The del(8)(?p12) and t(18;?) observed in the Gb a n d e d analyses were not obvious in the flow karyotype data.

Long In Vitro Culture. With c o n t i n u e d passage in culture, both the flow karyotype and G-banding data (Figs. 2C and 3B and Table 4) indicated that the chromosomal rearrangements selected near the point of immortalization were maintained. In addition, further selection of a number of additional chromosomal rearrangements was

240

c. l,. {;oolsl}v et al.

,l{ Z

F i g u r e 2 t,'l{}w karyolype analyses of HSI~'4-TI2 at 56, 136. zmd 31(1 t}{)l}ulati{}n doublings {PI)s} are shown iEI t}anP,ls A C, rest}{~{:|iv{%,'. Null]b{~rs ~l{,~xt |{} {}~t{;h i}{!ak l'{,~t}P{}S{}lll IH{! {;HroIllOSOlll(~,{s} {:o~]tain{~d in that t}~,ak. I}{}akslat}~l{}{twith M# ~l['{~al}norn]al peaks firs! i{len]tiIie,d al PI} 136 {i}an{!l B}. A{i{titi{}nal al}normaI i){,~aks are eviden! in t}an{~l C {]}l} 31(}}.

m

13

O O U

HOECHST 33258

o b s e r v e d . T h e o n l y significant loss of c h r o m o s o m a l material n o t e d was for the Y c h r o m o s o m e . In a d d i t i o n , one c o p y of c h r o m o s o m e 20 was f r e q u e n t l y lost in late passage HSF4-T12.

Tumor-Derived Cell Lines. Bivariate flow k a r y o t y p e analyses of HSF4-T12 TU2, H S F 4 - T 1 2 T U 4 , a n d H S F 4 - T 1 2 TU8 are s h o w n in Figure 4 A - C , r e s p e c t i v e l y , and the G - b a n d e d a n a l y s e s are s u m m a r i z e d in Table 5. T h e TU2, TU4, and TU8 cell lines w e r e e s t a b l i s h e d from r e c u l t u r i n g of i n d e p e n d e n t t u m o r s o b t a i n e d after i n j e c t i o n of H S F 4 - T 1 2 (PD z 136) into i m p l a n t e d G e l f o a m sponges in n u d e mice. Of the changes d e t e c t e d in the flow k a r y o t y p e of HSF4-T12 {PD = 136), - 6, - Y, and M3 [i(6p)] w e r e

241

M1

A

i

~

M~

'i! ¢,

M2

D _ _

i

q

F~

.

G . . . . . . . . . . . . .

11

B

M3

G

.

.

.

.

.

.

.

.

.

.

M2

x

Figure 3

Y

Representative G-banded karyotypes of HSF4-T12 at 136 [37,X, Y , - 2 , - 4 , - 5 , 11, 1 3 , - 1 4 , - 1 4 , 1 5 , - 2 0 , - 2 0 , - 2 2 , 3 q ' , 6 p ,6p ,18p ,+i(6pJ,+t(2p14q). +t(14q15q},t{21;?),+mar] and 263 [73,XX,+1,+1, 2 , + 3 , + 3 , + 4 , + 5 , + 5 , 6 , + 9 , + 1 0 . + 1 0 , + 1 1 , + 1 2 , 1 4 , - 1 4 , + 1 6 , + 1 7 , + 1 7 , 2 0 , - 2 0 , + 2 1 , + 2 1 , + 3 q + , + 3 q + , 6 p ,+i(6p),+i{6p], +i(7q),8p , + 1 8 p - , + t ( 2 p 1 4 q ) , + t ( 1 4 q 1 5 q ) , + t { 1 4 q l S q ) , + m a r l , + m a r 2 , + m a r 2 , + m a r 3 , + m a r 4 , + mar5, + mar6, + mar7] population doublings are shown in panels A and B, respectively. Chromosomes marked with M # are chromosomal rearrangements consistent with abnormal peaks observed in the flow karyotypes. International System for Human Cytogenetic Nomenclature designations are shown in Tables 4 and 5 and in the text.

242 Table4

c . L . Goolsby et al.

(;-banded karyotypeamdvses several PD l e v e l s

Pl)s

Loss"

53

Nolw

["l'(~q ( l e l l t:V

13(i

Y

')5/Z5

263

20 Y

23/23 23/23

for HSIr4-T12 at

RI!a r r a l l g ( ! l l l ( ! t l l s `J

l;r(!(ltl(!ll(:V

i(3:'f')((127':':':)) I(21)411l 1(2l)14(t] l (',k':))[(l27'(:'.e) i(611] (](,l(6)(pl 1.2) (hd[Sl(i)12'fl I[ 14qlSqt i] 15(I) tt 18:'~)(p':':?t l[2p14(I) l( 3 :'?]((127':':':'l i[1;])) (h,l(6H[)l 1.2] (lel(81l l)l 2':'] l( 14(llS(l) t( I ~k'?l{11'?:?] l"i~ (~ nmrkers

2[)/22 5/25 12/25 2(I/25 23/25 25/25 14:25 15:25 1025 ~725 21:23 23:23 23/23 22/23 2():23 23'23 I!1723 11:23

Alfl)rcviafions as in 'l'abh~ I. " I,()ss oi (IhI'I)IIIOSOIII(!F, ()[ chl'lill]i)NillllaI[ S(~Ol]l(!ll[S ilild ]'I!I]ITHII~[![]I[!]IIS [)l'(!S (!Ill ill 2{1(~) Ol' ]I!SS Ol th(', ( ' , - h a n d e d ( e l l s n m l l \ z(!(I is ii(l{ >4]l(l\Vll.

maintained in the flow" karyotypes of TU2, TU4, and TU8. The most striking feature in the flow karyotype data c o m m o n to TU2, TU4, and TU8 was the reduction in the number of counts in the normal chromosomes 13, 14, and 15 peaks. Although less obvious, there was also a significant reduction in the chromosome 20 peak volume. C h r o n l o s o l n e 14 a n d 15 m a t e r i a l was g e n e r a l l y a(:(:ounte(l for in the t(14q:l 5(i). f l o w ever. all t h e t m n o r s f r e q u e n t l y ha(l a n o r m a l 14 ( : h r o m o s o m ( : as well; 12 of 24, 20 of 20, an(l four of 21 in TU2, TIJ4, mM TIIS, resl)e(:tiv(,h~. G - b a n d e ( l (lata of all huu(ir(h:rive(l (:ell l i n e s s h o w e d fre(luen! telomeri(: a s s o c i a t i o n s b e t w e e n a lllllnber of (:hrolllOS()nl(;S, w i t h the m o s t fre(luenI b e i n g b(:tw(;en 12(I an(] a variety of o t h e r (:hromos o m e s . Specific a l t e r a t i o n s not pr(~sent in all (if l h e t m n o r cell lines are (lis(:usse(l below. t t S F 4 - T 1 2 TU2. T l l e M 1 p e a k , ( : o n s i s t e n t w i t h t h e t ( 2 p 4 q ) a n ( l t ( 2 p 1 4 ( 1 ) o b s e r w : ( t i n H S F 4 - T 1 2 (PD 1361, w a s p r e s e n t : h o w e v e r , o n l y t h e 1(2[);14(I) was evi(lent. Alt h o u g h t h e t ( 1 4 q l 5(i) w a s obs(~rved in 13 of 2(,)Tt!2 (;ells, the M2 peak w a s not p r e s e n t . T h i s t r a n s h ) c a t i o n m a y hay(; h a d s o m e s m a l l ( h : l e t i o n s r e l a t i v e to t h e o t h e r lines. T h i s m a y e x p l a i n t h e a b n o r m a l (:hromosom(! 7 peak it) tile flow k a r y o t y p e , whi(:h s h o w e d a s i g n i f i c a n t i n c r e a s e in tile n u m b e r of c o u n t s r e l a t i v e to o i h e r a(ljaceld a u t o s o l n a l l)eaks a n d w a s s o m e w h a t broa(lene(l. T h e 6q m a l e r i a l , [)reseut (is a (lislin(:l (:loual r e a r r a n g e m e n t d e l ( 6 ) { p l l . 2 ) in H S F 4 - T I 2 , was n()i observe(l; h()wever, an i(6(1) was o b s e r v e d ill 27 of 29 TU2 (:ells. In a(Idition, t h e M4 peak pr(;sent in H S F q - T 1 2 (PD 136) was m i s s i n g . H S F 4 - T 1 2 T U 4 . The G-banded data for HSF4-T12 TU4 was in agreement with the b i v a r i a t e flow k a r y o t y p e r e s u l t s w i t h the (',x(:epti(m of t w o n o r n m l - a p p e a r i n g 13 (:hrom o s o n m s in all T U 4 (:ells a n a l y z e d . T h e s e (tala w o u l d be most c o n s i s t e n t w i t h a n a l t e r a t i o n in a f r a c t i o n of t h e s e ( : h r ( m l o s o m e s not d e t e c t e d at t h i s level of [)an(ling that

K a r y o t y p e of a Tumorigeni(: H u m a n Cell Line

Z q) ).-

243

F i g u r e 4 Flow karyotype analyses of HSF4T12 TU2, HSF4-T12 TU4, and HSF4-T12 TU8 are shown in panels A-C, respectively, Numbers next to each peak represent the chromosome(s) (:ontained in that peak. Peaks labeled with M# are abnormal peaks.

O O

e~ -r U

HOECHST 33258 still might lea(t to a redu(:tion in the ilumber of coullts in the normal 13 (:hromos(mle peak. N e i t h e r the M1 peak in the bivariate karyotype nor t(2p4q) nor t(2p14q) was o b s e r v e d in TU4. M2, consistent w i t h the i(15) and t(14q15q) observe(/ in [lSIr4-T12 (PD 136), r e m a i n e d in TU4, but ()nl~, the t(14q15q) was noted. A n e w (:I(mal rearran~,ement, t(3:3) (q28 or 29:1)12 or 13). was observe(t in 18 of 20 TI+I4 (:ells. t t S F 4 - T 1 2 T[!¢L All T118 (:ells c o n t a i n e d either o n l y a normal 8 c h r o m o s o n ) e or two) normal 8 c h r o n l o s o m e s and a del(8)(t)127). The M1 peak was retaine, d, but again o n l y the t(2p:14q) was noted. M2 was retained, but o n l y the t{14q;15q) was observed. T w o n e w (:lonal rearrangements, t(1(,);?}(1~13.3;?) and i(gp) w e r e observe(t, and two n e w

244

c . L . Goolsby et al.

Table 5

G-banded karyotype analyses for three HSF4-T12 tumor derived cell lines

Cell line

Loss"

Frequency

HSF4-T12 TU2

-13 15 20 -Y

6/24 5/24 18/24 24/24

HSF4-T12 TU4

15 2(/ Y

12/2/) 12/2(I

-13 -15 -20 -Y

21/21 21/21 21/21 21/21

-

HSF4-T12 TU8

5/20

" Loss of chl'olllO SOl l l (, o r c h r o l / l O s i m l a l

Rearrangements" t(2p14q} t{3;?]{q27?;?} i(6p} t(14q15q} t{lS;?J(p?;?) t[3;3){q28or 29; p12 or 13) i{6t}}

t{14q15q} t(18;?}{p?;?) t{2pl4q} t{3;?}(q277;?} del{6}{pl1.2) i(6p] del{8){p127} i(9p) t(14(l15q) t(18;?){p?;?) t{19:?]{|}13.3:?}

Frequency 14/24 23/24 23/24 24/24 18/24 18/20 4/2(}

4/2(} 5/20 21/21 21/21 21/21 21/21 11/21 20/21 19/21 21/21 21/21

s{}~lllOlltS tllli] r(~IFFtlII~IHIIIHIts pF(!S{!l|[ In ")()[~'c}of" h!ss ()] t h .

Gq}anded cells analyzed is not shown.

peaks in the flow karyotype of TU8, M5 and M6, were consistent with these changes, respectively (Fig. 4C}. Thus, although some new rearrangements were observed and some rearrangements present in HSF4-T12 {PD - 136} were retained, a characteristic common to all the tumors was loss of chromosomal material.

DISCUSSION

In this study, the pSV3-neo plasmid was used to transfect a strain of normal HSF4. This initiated a multistep neoplastic process that ultimately resulted in a {:ell population with all of the cardinal characteristics of a neoplastic population (ie, immortal, transformed, abnormal karyotype, and tumorigenic). Although many studies have shown that T-antigen expression is required for m a i n t e n a n c e of the transformed phenotype [31-34], SV40-infected mouse 3T3 cells randomly cloned without selective pressure exhibited a wide range of phenotypes from minimally transformed to maximally transformed [35]. Our results clearly show that expression of T antigen alone is insufficient for expression of most in vitro transformation markers in SV40transformed h u m a n fibroblasts. Immediately after transfection, the only significant change in cellular properties was a reduced dependence on serum for growth. After expression of T antigen in 100% of the {:ells in the population, extensive passage in culture was necessary for acquisition of and progression in the expression of these in vitro transformation markers. This progression of neoplastic phenotypes is similar to some reports of events observed after viral infection of h u m a n {;ells [11,361. Furthermore, on a cellular basis, immortalization of h u m a n fibroblasts after transfection with plasmids containing the early genes of SV40 is a rare event [371 and does not occur as a direct result of T-antigen expression. HSF4-T12 cells were 100% T-antigen positive many passages before crisis and immortalization. In HSF4-T12, early passages after transfection were characterized by nonspecific chromosomal changes, with every cell exhibiting a nearly u n i q u e karyotype. At this stage, before immortalization or significant transformation, only a subtle t(3;?} re-

Karyotype of a Tumorigenic Human Cell Line

245

arrangement was observed in a significant proportion (20 of 22) of tbe cells, and the effects observed can be considered r a d i o m i m e t i c and scored as c h r o m o s o m e aberrations such as gaps, breaks, dicentrics, and fragments [38]. The dicentrics observed occasionally resulted from telomeric associations between the chromosomes. Because of the large number of changes observed, the bivariate flow karyotype results for these early passages of HSF4-T12 were particularly useful in demonstrating the lack of consistent rearrangements within the population. Some of this variability in karyotype might have occurred because HSF4-T12 was not e x p a n d e d from a single resistant colony. This appears not to be the case. Other cell strains examined in this study (data not shown) and in our other studies that were e x p a n d e d from single colonies [39] exhibited similar or greater (:hromosomal variability. In addition, limited examination of several subclones isolated from early passage HSF4-T12 {data not shown) all showed karyotypic instability. Although a similar picture of early chromosomal changes has been reported after either viral infection [11, 16, 18-20I or transfected early genes [40], most investigators have not c o n c l u d e d that they played any important role in the oncogenicity of SV40. In the case of active virus, the semi permissive nature of h u m a n cells suggested that the clastogenicity was the result of cytopathic effects. In the case of plasmid constructs, most reports known to us haw ~,used constru(:ts, such as pSV3-neo used in this study, that have SV40 origin sequen(:es an(t support autonomous replication of plasmid material 141], which has been considered responsible for the early clastogenicity [40]. We confirmed the presen(:e of this material in all seven early transfe(:tants tested (four from HSF4 (data not shown) and three reported in ref. 39). We have also shown, however, that a similar pi(:ture of kary()typi(: instability follows transfection with p l a s m i d s not possessing ori sequen(:es and not produ(:ing extrachromosouml T-antigen sequences. These data differ marke(lly from a recent speculation of Donahue and Stein [42[. Just after immortalization, consistent chromosomal rearrangements were observed, which probably reflected in part the clonal selection of an immortalized t)opnlation at senescence. In addition, attenuation of the clastogeni(: phase was probably involve(t. Flow karyntype analysis was useful in verifying which of the many rearrangements observed in HSF4-T12 and in HSF4-T12 tumor-derived cell lines were represented in a significant proportion of the cells. The M4 rearrangement whi(:b was (:learly represented in the flow karyotype, was not identified in the G-banding analyses. The t(2p:4q) and t(2p;14q) (M1) are of interest because the breakpoint on both is at 2 p l 1-13, the location of the tumor growth factor-(~ (TGlr-c~) gene I43]. Whether this has resulted in any rearrangements of the TGIV-(~gene or changes in the expressinn of TGF-(~ is currently being investigated. With very long in vitro passage (more than 230 PDs), additional rearrangements were. sele(:ted in HSlV4-T12. In general, the rearrangements observed were balanced, without the detectable loss of any chromosomal material. This picture is similar to that reported for the SV40 transformation ()f a uroepithelial cell strain [16]; however, it is different from the cases reporte(t by Hoffschir et al. [15] for a number of SV40-transformed h u m a n fibroblasl lines in whi(:h loss and deletions were common. Hoffschir et al. [15] also con(:luded, based on indirect evidence, that most rearrangements occurred after tetraploidization. In the present study, the chromosomal rearrangements were probably established before the p o p u l a t i o n was tetraploid. On the other hand, the tumor-derived cell lines (HSF4-T12 TU2, TU4, and T[J8) were characterized by unbalanced changes and frequently exhibited loss of chronn)somal material. Although some rearrangements present in HSF4-T12 at the time cells were injected into n u d e mice were retained, new balanced chromosomal rearrangements were not frequently selected during the in vivo growth of tumors in nude mice. The discovery of tumor suppressor genes and the involvement of their loss or mutation in h u m a n tumors has drawn attention to the role of deletions and loss of heterozygosity [44-49]. The frequent loss of c h r o m o s o m e 13 and the presence of i(6p), which was

246

¢:. L. (;oolsby et al.

observed in TU2 and TIJS, is strikingly similar to the pattern of (:bron]osomal ~:bange o b s e r v e d in r e t i n o b l a s t o m a i50 52I. W h e t h e r loss or m u t a t i o n of r e t i n o b l a s t o m a , i)53 or other suspe(:ted tun)or s u p p r e s s o r gene sequenl;es has o(:(:urred ill the tuml)rd e r i v e d cell lines is c u r r e n t l y u n d e r investigation. TU4 e x h i b i t e d a t(3:3)((12~ ()r 29:1)12 or 13) rearrangement. A d e l e t i o n near this region of 3p has been r e p o r t e d in small (;ell lung (:art:inoma 19, 53] and in renal cell car(:inoma I9.54]. T w o of the t u n ) o r - d e r i v e d cell lines s h o w e d loss ()f the 6(t material. In a d d i t i o n , all tumor-(teriv(;(t (:ell lines s h o w e d freque, nt telomeri(: asso(:iatiol)s t)et w e e n 12q and a xminber i)f other (:hrom(tsomes. T h e s e types of telomeri(: asso(:iatil)ns []ave l]een reported by Ni(:bols et al. 1551 it] the SV40 lral]sl/)rmatiol] ttf I m m a n fibroblasts, a n d telon]eri(; asso(:iatiol)s i n v o l v i n g 12(t were ret:tmtl,v rt,'porte(t in squan]ous cell carl:inoma 1561. In con(:lusion, the i m m o r t a l i z a t i o l ] and in vitro transft)rlnation (if 11SE4-T12 after transfel:tion w i t h the early genes of SV4(] was at:(:ompanie(I [)y (:h)nal selection of a n u m b e r ()f b a l a n c e d (;hr/)mosomaI r e a r r a n g e m e n t s from a (:ell p o p u l a t i o n g e n e r a t i n g n u m e r o t t s nol]spe(:ific c:hron]osomal (:banges. T h e f o r m a t i o n (If tumors in nu(le mi(:e by I t S E 4 - T I 2 cells was (:hara(:terize(I I)y loss of (:hrolnoson]al material, howeww. A similar pattern of (:hroml)somal c h a n g e was (xbserve(I in the SV40 t r a n s f o r m a t i o n of u r o e p i t h e l i a l (:ells ill whi(:h the tumorigeni(: l)henotyl)e was i[l(hxl',ed by (:hemi(:al t r e a t m e n t [16, 171. We propose, that the r e a r r a n g e m e n t s ol)scrved d u r i n g in \ i t r o and it) v i v o progression (if H S F 4 - T I 2 are n/)l u n i q u e to the SV40 trax)sformalion t)rt)cess, but that T-antigen i n d u c e d the karyotypit; (genomit:) instability leading to rearrangen)ents from w h i c h a [)umber w e r e sele(:ted base,(I on the sele(:tive ltressure that our system i m p o s e d on the (',ells.

This w o r k was SUpl/orted in part bv lt]~: ttnited Slates I)oparllll(!Ill o[ l:,]mrgy, lh~ I,t)s :\]alnt),~ H o w (]ytomelry Resour~;e hv Nltt (;ram No. RR01;:115, NIH P,t~sear~:h l:ellowshit~ A w a r d No. 5 1;32 (]A07910. N l l t (]r;ml No. RR05:171). im(I tile V[]t(!l'a[ls Admit]iMralicm (i'vlt~di(:al I'~t~seaI't:h Servicel.

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