cancer res 1988 pasquale 2715 9

7/25/2019 Cancer Res 1988 Pasquale 2715 9

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[ CA N CER R ES EA RC H 4 8, 2 71 5- 27 19 , M a y 1 5, 1 98 8]

Tumorigen ic ity and Oncogene Exp re ss ion in Ped ia trie Cance rs1

S teven R . P asqu ale,2 G ary R . Jon es,3 C laus-Jen s D oersen , an d B ern ard E . W eissm an4

D ivision of H em atology-O ncology, Childre s H ospital of Los A ngeles and D epartm ent of M icrobiolo gy, U SC School of M edicine [S. R . P ., G .R .J., B.E. W .],

L os A n ge le s, C a li fo rn ia 9 00 3 3, a nd C o rp or at e R es ea rc h D iv is io n, P ro ct er a n d G a m bl e C o mp an y, M i am i V a ll ey L ab or at or ie s, C in ci nn at i, O h io 4 52 39 -8 70 7 [ C- J. D .J

ABSTRACT

C yto gen etic a nd ep id em io l g ica stu dies o f p ed ia trie ca ncer s h av e

implicated a loss of genetic inform ation in the development of these

tum ors. In contrast, other studies have show n that activation of endoge

nous oncogenes is a com mon event in these cancer cells. The technique

of somatic cell hybridization provides a model for investigating the

interaction betw een loss of genetic elem ents and onco gene activation in

pediatrie cancers. A variety of hum an-hum an cell hy brids w ere form ed

b etw een a tu mo rig en ic a du lt ca rcin om a a nd rep resen ta tiv e tu mo rig en ic

pediatrie cell lines. All hybrid cells w ere com pletely suppressed for

tumor-form ing ability when assayed in nu/nu (nude) m ice. W hen the

expression of the N -m yc, c-m yc, and sis oncogenes and tum origenicity

were examined in the same hybrid cells, no correlation was found,

suggesting that the expression of these oncogenes in these hybrid cells

did not appear to be controlled by putative tum or suppressor genes.

Thus, tum origenicity behaves as a recessive genetic trait in pediatrie

c an ce rs . F ur th er mo re , d iffe re nt g en et ic e le me nt s m ay b e lo st d ur in g tu mo r

d ev elo pm en t o f a du lt ca ncers a s o pp osed to p ed ia trie ca ncers .

INTRODUCTION

The development of neoplasia in human cells appears to

in vo lv e a n umbe r o f g en etic a lte ra tio ns in clu din g th e a ctiv atio n

of endogenous oncogenes and a loss of norm al genetic infor

m ation (1,2). W hile the presence of altered oncogene expres

sion has been well documented in human tumor cells, the

co ncep t o f a lo ss o f no rm al g enetic elem en ts in th e d ev elo pm en t

of hum an cancers is based on evidence w hich has been inferred

from several types of studies. S everal pediatrie cancers have a

rare hered itary fo rm w here deletio ns in sp ecific ch ro moso mes

h av e b ee n a sso cia te d w ith th e e tio lo gy o f th e d is ea se . E xamp le s

include the deletion in chrom osom e 13ql4 observed in retino-

blastoma (3) and the deletion in chromosome 11pi3 that is

seen in W ilm s' tum ors (4). A deletion in the p arm of chrom o

som e 1 in neuroblastom as has also been observed, although a

hereditary form has not been reported (5). It w ould appear that

th e lo ss o f n orm al g en etic in fo rm atio n p re se nt in th es e d ele tio ns

is associated w ith th e m align an t sta te.

A nother series of studies supporting a loss of genetic infor

m ation in the developm ent of hum an cancer involves the char

acterization of som atic cell hybrids. S om atic cell hybrids be

tween human tumor cell lines and normal human cells are

in itially sup pressed for tum orig en ic p oten tial (6 -9 ). U pon co n

tinued passage in culture, tum origenic variants appear w hich

have lost specific chrom osom es. In the case of H eL a x norm al

h um an fibro blasts, co pies o f b oth ch ro mo so mes 1 1 a nd 1 4 w ere

lost upon reexpression of tum origenic potential (10, 11). In

hybrids betw een H T 1080 and norm al hum an fibroblasts, loss

of chrom osom es 1 and 4 was associated with reexpression of

Rece iv ed 10/ 8/ 87 ; r ev is ed 1 /2 1/ 88 ; a cc ept ed 2 /1 9/ 88 .

T he c osts o f p ub lic atio n o f th is a rtic le w ere d efr ay ed in p ar t b y th e p ay me nt

o f p ag e c ha rg es. T his a rtic le m ust th ere fo re b e h er eb y m ar ke d a dv er tis em en t in

a cc or da nc e w ith 1 8 U .S .C . S ec ti on 1 73 4 s ol ely to in di ca te t his f ac t.

1 Th is w or k w as f un de d b y U S PH S G ra nt S 07 RR 05 46 9-2 3 fro m th e N atio na l

C an ce r I ns tit ute a nd b y t he C la yt on F ou nd at io n Mo le cu la r B io lo gy P ro gr am .

2 S up po rte d b y U SP HS T ra in in g G ra nt 2 T3 2A I0 70 78 -0 7 fr om th e N IH .

3 P r es en t a dd re ss : C h il dr en s H o sp it al R es ea rc h F o un da ti on , C in ci nn at i, O H

45229.

T o w ho m r eq ue sts fo r r ep rin ts s ho uld b e a dd re ss ed .

tum origenicity in the hybrid cells (8). A n analysis of R FL Ps5

in the HeLa x fibroblast hybrids showed that only the chro

mosome 11 from the normal fibroblast parent was lost upon

re exp ressio n of tu mo rig en icity (1 1, 1 2). T hu s, it ap peared th at

norm al genetic elem ents present on these chrom osom es have

either been lost from these tum or cells or are no longer func

tional.

Previous research in the area of somatic cell hybrids has

p rim arily in vo lv ed cell lines d eriv ed from ad ult tu mo rs. S in ce

c yto ge ne tic a nd familia l stu die s h av e im plic ate d a lo ss o f n orm al

g ene tic info rm atio n from p ed iatrie ca nce rs, it w as of intere st to

d ete rm in e th e g en etic b eh av io r o f tumo rig en ic ity in th ese tumo r

cells (3-5 ). T herefore, a v ariety o f cell h yb rids b etw een an ad ult

carcin om a, H eL a, an d p ed iatrie ca ncer cell lin es w ere iso lated .

T um origen ic ity has b een sh ow n to b eha ve as a re cessiv e g ene tic

trait in H eL a cells, th e pu ta tiv e tum or sup presso r ge ne(s) b ein g

asso ciated w ith ch ro moso me 11 (13 ). H eL a w as used to p rev en t

problem s of nonviability observed in hybrid cells betw een hu

m an tum or and norm al hum an cells (14).

T he data in this report dem onstrate that tum origenicity be

haves as a recessive genetic trait in pediatrie tum or cells. Fur

therm ore, it appears that different genetic elem ents control

tum origenic expression in pediatrie and adult hum an tum or

cells. In addition, it is show n that the expression of the N -m yc

and c-m yc oncogenes, w hose overexpression has been associ

ated w ith the developm ent of several pediatrie cancers, does

not correlate with the tum origenic potential of these hybrid

cells. F ina lly , th e ex pressio n of th e sis o ncog en e, w hich c od es

for the P DG F, does not correlate w ith tum origenicity in these

cells.

M ATERIALS AND M ETH OD S

P arent and H ybrid C ell L ines. T he hum an cell lines utilized in this

stu dy a re lis te d in T ab le 1 . A l l ce ll lin es h av e b een p rev io usly c hara c

te riz ed a nd are c ons isten t w ith th e ty pe o f tu mo r fro m w hich th ey w ere

d er iv ed . C ell l in es w er e g rown i n e it he r D ul be cc o's m od if ie d E ag le 's o r

R PM I 1 64 0 s up plem en te d w ith 1 0% fe ta l ca lf seru m (J. R . S cien tific,

W oodland, C A). A ll cultures w ere routinely tested for M ycoplasm a

co nta min atio n b y D AP I assa y (1 5) a nd w ere n eg ative . P ro duc tio n a nd

selection of hybrids were accomplished by the HAT and ouabain

s el ec tio n m et ho d r ep or te d e ls ewhe re ( 16 ). V ar io us c ombi na ti on s w er e

m ad e p os si bl e b y t he u se o f t he u niv er sa l f us er c el l l in e D98OR , de riv ed

fro m H eL a, w hich is b oth h yp ox an th ine p hos ph orib osy l tra nsfera se

d ef ic ie nt a nd ouaba in r es is ta nt ( 16 ).

H yb rid Ana ly se s. H yb ri d f orma tio n w as c on fi rm ed b y c hr omos ome

analysis. C hrom osom es w ere prepared according to the m ethod of

N els on R ees e t at., an d at lea st 2 5 rn cta ph u.su sp rea ds w ere ex am in ed

for each clone (17). In cases where chromosom e number was not

inform ative, hybrids w ere also confirm ed by R FL P analyses using

probes for specific chrom osom es. D NA probes specific for chrom o

so me s 1 1 a nd 1 3 w ere p ro vid ed b y D r. E . S riv atsa n of th e D epa rtm en t

o f O ph th alm olo gy at C hild re n's H osp ita l o f L os A ng ele s. R FL P an al

y se s w er e p er fo rm ed a s p re vio us ly d es cr ib ed (1 2) .

T um origenicity A ssays. C ell suspensions containing 2 x 10' to 2 x

1 0* c ells/0 .2 m l w ere in jec te d s .c. into th e ce ntral m id lin e o f c ong en i

tally a th ym ic n u/n u m ice . R etin ob la sto ma h yb rid s w ere also in jec te d

'T in 1 a bb re via tio ns u se d a re : R FL P, re stric tio n f ra gm en t le ng th p oly mo r

p hi sm ; PDGF , p la te le t- de ri ve d g rowt h f ac to r, cDNA , c ompl eme nta ry DNA.

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T UM O RI GE NI CI TY N D O NC O GE NE E XP RE SSI ON I N C EL L H Y R ID S

5 2 kb

2 2 k b

2 jO k b

F ig. 1. E xpression of the N -m rc onco gene in pare ntal and hybrid cells. T he

e xp re ss io n o f th e N -m>>co nc og en e in th e h yb ri d c ell l in es b etw ee n H eL a (D9 8 )

a nd a n eu ro bla stoma (LA -N -S ) a nd a r eti no bla stoma (Y7 9) is s hown . T he e xp re s

s io n w as d ete rm in ed b y N or th er n b lo ttin g te ch niq ue s a s o utlin ed in M ate ria ls

and M ethods. Each lane contains 4 M n of total R N A. H Hyl7P2C TuO is a

t umo r gen ieegr eg an d er iv ed f rom t he D98OI>X Y79 hyb ri d c el l l in e HHy I7P2 .

T o c on firm e qu al lo ad in g o f R NA , th e f ilte r w as su bs eq ue ntly h yb rid iz ed w ith ap ro be for a -t ubul in ( lower por ti on o f f ig ur e) , k b, k il ob as e.

I I I M I

2 2b

F ig . 2 . E xp re ssio n o f th e c -m yc o nc og en e in th e p are nta l a nd h yb rid c ell lin es.

T he expression of the c-m yc onc ogene w as determ ined by N orthern blotting

t echn iq ue s a s d es cr ib ed i n Ma te ri al s and Me thod s. The p ar en ta l c el l l in es HeLa

(D 98 OR ), tw o W ilm s' tu mo r c ell lin es ( G4 0I a nd S K-N ep -l), a nd th e h yb rid c ells

a re s hown . E ac h l an e c on ta in s 4 0 igf to ta l c el lu la r RNA. k h. k il ob as e.

was not observed in the other tw o hybrid cells. Thus, the level

of expression of the c-myc oncogene did not correlate with

tumo rig en ic p ote ntia l in th e h yb rid c ells .

E xpression of the sis/P DG F-2 oncogene has been observed

in many human tumor cells and has been associated with the

production of a growth factor activity (25, 26). The W ilms'

tum or cell line, G 401 , e xp ressed de tectab le lev els of m RN A fo r

this oncogene. Expression of the sw /PDGF-2 oncogene has

been o bse rv ed in an othe r k id ney carcino ma (2 7). W e th erefo re

fe lt it w ou ld b e o f in te re st to d ete rm in e w he th er a ny c orre la tio n

existed betw een tum origenic potential and the expression of

this oncogene. F ig. 3 show s the expression of the sis oncogene

in this set of cell hybrids. The HeLa x W ilms' tumor hybrids

show ed variable levels of expression of 5/5 as com pared to the

W ilms' tumor parent; HHylSPl showed no expression of the

.s n co gen e, w hile H Hy 1 4 sho wed th e sam e lev els as th e G 40 1

parent. A third clone HHyl5P3 showed a somewhat reduced

level of m RN A w hen com pared to the parent. S ince H HylS Pl

a nd HHy 1 4 a re b oth n on tumo rig en ic c ell lin es, tumo rig en ic ity

d id n ot co rrelate w ith \/.v o ncog en e ex pressio n in these ce lls.

DISCUSSION

W hile p rev io us stud ies fro m seve ral la bo rato ries h av e dem

onstrated that tum origenicity behaved as a recessive genetic

trait in tumor cells derived from adult cancers, the genetic

behavior of tum origenicity in pediatrie cancers had not been

investigated. T he im portance of determ ining this w as under

scored by the fact that m uch of the evidence w hich supported

th e ex isten ce o f recessiv e can cer g en es ca me fro m fam ilia l and

cy to gene tic stu dies o f p ed iatrie can cers. T he d ata in th is rep ort

d emon stra te th at tumo rig en ic ity b eh av es a s a re ce ssiv e g en etic

trait in cell lines derived from a variety of pediatrie cancers.

T hus, the cells have presum ably lost genetic m aterial w hich is

in vo lv ed in th e e xp re ssio n o f th e tumo rig en ic p he no ty pe . T he se

resu lts raise ob vio us qu estio ns ab out th e asso ciation be tw een

tumo r s up pre ss or g en es a nd c hromo soma l d ele tio ns th at h av e

b ee n a ss oc ia te d w ith c erta in p ed ia trie c an ce rs.

S ax on < /l. and W eissm an et al. have recently reported that

the introduction of a norm al hum an chrom osom e 11 into both

H eL a a nd W ilm s' tumo r c ell lin es s up pre ss es th eir tumo rig en ic

p ote ntia l (1 3,2 4). T he re fo re , th e p oss ib ility a ro se th at th e s am e

sion of the N -m yc oncogene m ight be correlated w ith tum ori-

genie expression in these hybrid cells. In order for this associ

ation to be consistent, reexpression of tum origenicity in the

hybrid cells should be accom panied by an increase in the levels

of N -m yc m RN A. A s show n in F ig. 1, there w as not a detectable

level of N-myc mRNA in the HHyl7P2-TuO cell line. Thus,

th ere d id n ot ap pear to b e a correlatio n b etw een th e sup pressio n

o f tu mo rig enicity in th ese h yb rid cells an d th e ex pression o f th e

N-myc oncogene .

Enhanced expression of the c-myc oncogene has been re

ported to occur in som e W ilm s' tum ors including the G 401 cell

line (23, 24). W e therefore exam ined the level of expression of

this oncogene in the H eL a x W ilm s' tum or cell lines. A s show n

in Fig. 2, while both of the W ilm s' tum or cell lines expressed

m oderate levels of c-m yc, H eL a cells show ed a high level of c-

m yc expression n tw o nontum origenic cells there w as a high

level of c-myc mRNA, while a third hybrid cell expressed a

level sim ilar to the W ilm s' tum or parent. The reduction in the

level of this myc family oncogene in one hybrid cell line was

sim ilar to that observed with the expression of the N-inyc

o nco ge ne in the Y 79 x H eL a hy brid cells. H ow ev er, th is ch an ge

kb

2 2 kb

F ig. 3. E xpression of the sis oncogene in the parenta l and hybrid cell lines.

T he e xp re ssio n o f th e .v i'.vn co ge ne w as d ete rm in ed b y N orth ern b lo ttin g te ch

n iq ue s a s out li ned i n Ma te ri al s and Me thod s. Expr es si on i n hyb ri d c el ls b etween

H eL a ( I > '> )n d a W ilm s' t umo r c ell l in e (G4 01 ) is s hown . E ac h l an e c on ta in s

4 0 f ig o f to ta l c ellu la r R NA . G 40 I.2 , a G 40 1 c ell lin e in to w hic h a n or ma l h um an

c hro mo so me 1 1 h a s b ee n in tro du ce d b y m ic ro ce ll tra nsf er, h as th e sa me le ve l o f

e xp re ss io n a s th e G 40 1 p ar en ta l c el l li ne . T he c on tr ol l an e c on ta in s t ota l c ell ula r

RNA f rom a g li ob la stoma c ell l in e (A 1 72 ). k b, k ilo ba se .

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T UM ORIG EN IC IT Y A ND O NCO GE NE E XPR ES SIO N IN C EL L H YB RID S

genetic element on chromosome 11 that is lost during the

development of W ilms' tumor is also absent in HeLa cells.

H ow ev er, th e da ta in th is stu dy c learly d em on strate th at h yb rid

cells betw een tw o different W ilm s' tum or cell lines and a H eL a

cell line are suppressed for tum origenic potential. T his result

im plie s th at W ilm s' tumo r c ells a nd H eL a c ells c on ta in d iffe re nt

g en etic le sio ns w hic h a re in vo lv ed in th e c on tro l o f tumo rig en ic

potential. F urtherm ore, because existing data w ould suggest

that W ilm s' tum or cells contain tw o copies of a genetic defect

a t ch ro moso me 1 Ip l 3 , these resu lts w ou ld su gg est th at th is site

is probably not the location of the H eL a suppressor gene.

Recently a cD NA clone has been identified as coding for the

p utative retin ob lasto ma (rb -1) g en e (2 8-30 ). T he ev id en ce fo r

this conclusion comes from the mapping of this sequence to

the area of chromosome 13ql4 as well as the absence of the

m RN A in retinoblastom as and osteosarcom as. T he data from

th ese c ell h yb rid iz atio n e xp erim en ts in dic ate th at tumo rig en ic -

ity can b e su pp resse d in a retin ob lastom a cell lin e (Y 79 ) as w ell

as an osteosarcoma cell line (QHS P16T) by the addition of

presum ably norm al genetic m aterial. Both of these cell lines

have been shown to have altered expression of the rb-1 gene

(28 -3 0). It w ill b e im po rtant to de term in e w heth er th e ad dition

of the norm al rb-1 gene to these cells w ill result in the abroga

tio n o f th eir tumo rig en ic p ote ntia l.

Wh ile th e e xiste nc e o f re ce ssiv e c an ce r g en es re sts o n c irc um

sta ntia l e vid en ce , o nc og en es h av e b ee n w ell c ha ra cte riz ed . S ur

p risin gly , little in fo rm atio n h as b ee n re po rte d o n th e e xp re ss io n

o f o nco gene s in som atic cell hy brid s. H yb rid s b etw een n orm al

human fibroblasts and two tumor lines having activated ras

g en es (8 , 9 ) w ere to tally su ppressed fo r tum orig en ic p otential

yet continued to express the M , 21,000 protein. T he influence

of other oncogenes on the tumorigenicity of hybrids has not

been r eport ed .

A ltered expression of the N -m yc oncogene has been associ

ated with several types of pediatrie cancers. W hen either a

n euro blasto ma cell lin e (L A-N -5 ) o r a retin ob la sto ma cell lin e

(Y 79 ) w hic h c on ta in ed amp lifie d c op ie s o f th e N -myc o nc og en e

was fused to a HeLa cell derivative which did not express N-

myc the hybri d cel ls w ere suppressed for tumori geni c potenti al .

In a concomitant manner, N-/nyc expression was also sup

p re ssed, d ue to th e lo ss of a mplificatio n o f th e N -/n yc o nc og en e

in th e h yb rid c ells. S in ce a ll h yb rid c ells a ro se from in de pe nd en t

fusion events, these results im ply a strong selective pressure

against the am plification of the N - wyr oncogene in the hybrid

cells. W hen tum origenicity w as reexpressed in the H eL a x Y 79

hybrid cells, there was no increase in the levels of N-myc

m RN A. T his resu lt rules o ut th e n ecessity fo r o verex pressio n

of the N -m yc oncogene in order for tum origenic potential to be

reexpressed.

W hen the expression of the c-m yc oncogene was exam ined

in the HeLa x W ilms' tumor hybrid cells, no correlation of

e xp re ss io n w ith tumo rig en ic p ote ntia l w as o bse rv ed , a lth ou gh

all parental cells expressed detectable levels of this gene. A n

exam ination of the am ount of c-m yc protein in the hybrid cells

w ill also be required to confirm phase results. It has been

proposed that N-myc and c-myc may control each other's

expression, making it unlikely that both oncogenes w ill be

expressed in the cell line (23). This m ight explain the strong

selection against the expression of the N -m yc oncogene in the

retinoblastom a and neuroblastom a x HeLa hybrid cells. W e

a re c urre ntly p ursu in g th is q ue stio n.

A utocrine production of grow th factors has been suggested

to play a role in tum or developm ent. P roduction of PD GF-like

factors by cells w hich overexpress the sis oncogene m ay be one

ex am ple o f th is m ech an ism . E xpression o f th e sis o nc og en e d id

n ot c orre la te w ith tumo rig en ic p ote ntia l in h yb rid c ells b etw ee n

a W ilm s' tum or cell line and H eia. In tw o hybrid cells, the level

o f ex pression o f the .v i.vo ncog en e w as sim ilar to th at o bserv ed

in the parental cell line. T his result agrees w ith previous data

on m icrocell hybrids w ith the sam e W ilm s' tum or cell line and

hum an chrom osom es t(X;l I) and t(X ;13) (24). H ow ever, it is

interesting to note that, in one hybrid cell line, sis expression

w as not observed. W hether this is due to a loss of an activating

factor or the ad ditio n o f a reg ulato ry g en e(s) is cu rren tly b ein g

investigated.

T he fo rm al p roo f of th e ex isten ce of rec essiv e c ance r an d/or

tumo r s up pre ss or g en es w ill re qu ire th eir iso la tio n b y mole cu la r

biological techniques. The data in this report, as well as in a

prev io us stu dy , w ould sug gest th e ex isten ce o f m ultiple tum or

suppressor genes in the hum an genom e (31). Introduction of

in di vi du al c hromosome s by m ic ro ce ll h yb ri di za tio n may fu rth er

define the num ber of different genes w hich are responsible for

the control of tum origenic expression in cancer cells (13, 24,

32). H ow ever, the hybrid cell lines generated in this study w ill

be useful in identifying protein or oncogene changes which

co rrelate w ith tu mo rig en icity . In th is m an ner, va lua ble in for

m ation about the functions or products of putative recessive

can cer g enes m ay b e g ain ed .

KNOW L EDGMEN T S

W e w ish to thank D r. Jayne K linger, R uth O chiai, and John Chou

fo r th eir assistan ce o n m an y a sp ec ts o f th is pro jec t. W e a lso th an k D r.

S tuart A aronson, D r. W illiam B enedict, D r. E ric S tanbridge, D r. O .

F od sta d, an d D r. E mil B oge nm ann for th eir g en erou s gifts o f p are nta l

cell lines and appropriate oncogene probes, as w ell as their advice.

A dd ition ally , w e th an k D r. K eith R ob bin s, D r. H isan og a Ig era sh i, an d

Alan M ock for instructing us on the finer points of Northern blot

analysis. M arge G reen, M ary Longo, and Janet Peterson provided

tec hn ica l ass istan ce. P atric e Jo hn sto n a nd C urtis H all ass isted in th e

p re pa ra ti on o f t he manu sc ri pt .

REFEREN ES

1 . A rm ita ge , P ., a nd D oll, R . A tw o-sta ge th eo ry o f c ar cin og en esis in r ela tio n

to th e a ge d istrib utio n o f h um an c an ce r. B r. J. C an ce r, //: 1 61 , 1 95 4.

2. B ishop, J. M . T he m ole cular ge netics of cancer. S cie nce (W ash. D C), 235:

3 05 -3 11 , 1 98 7.

3. K nudson, A . G ., Jr. R etino blastom a: a prototypic heredita ry neopla sm .

S em in . O nc ol. , 5 : 5 7- 60 , 1 97 8.

4 . R ic ca rd i, V . M ., S uja nsk y, l ., S mith , A . ( .'.. a nd F ra nc ke , U . C hro mo so ma l

imba lance i n t he An ir id ia -W i lms ' t umo r a ss oc ia ti on : 1 I p i nt er st it ia l d el et io n.

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1988;48:2715-2719.Cancer Res

Steven R. Pasquale, Gary R. Jones, Claus-Jens Doersen, et al.

Tumorigenicity and Oncogene Expression in Pediatric Cancers

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