mechanism of h-ras oncogene activation in mouse squamous carcinoma induced

7/25/2019 Mechanism of H-ras Oncogene Activation in Mouse Squamous Carcinoma Induced

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[ CA N CER R ES EA RC H 4 8. 5 56 -5 58 , F eb ru ar y 1 , 1 98 8]

Mechanism of H -ras Oncogene Activation in Mouse S quamous C arcinoma Induced

by an A lkylatin g Agen t1

A nne E. H ochw alt, Jerom e J. Solom on, and Seym our J. G arte2

N ew Y or k U niv er sit y M ed ic al C en te r, In sti tu te o f E nv ir on me nta l M ed ic in e, N ew Y or k, N ew Y or k 1 00 16

ABSTRACT

A mous e s ki n s qu amous c el l c ar ci noma induc ed by top ic al a pp li ca ti on

o f the d irec t -ac ting a lky la ting agen t /3 -p rop io lac tone contain s an ac tiva ted

11- ra v on cogene w i th a n ATr an sv er si on a t th e s ec on d n uc le oti de o f

c odon 61. Th e mu ta ti on wa s d et ec te d i n N IH3T3 t ra ns fr et an a nd o ri gi na l

tumor DNA b y a n Xha l r es tr ic tio n e nz yme p ol ymor ph ism a nd c on firme d

by o ligonucleo tide misma tch hyb r id iza tion. The mutat ion was no t seen

in th e liv er o f th e sa me a nim al. T he a ctiva te d on cog en e also ex hib ite d

s ev er al r es tr ic ti on e nzyme polymo rphi sms i n t ra ns fr et an DNA due to a

reciprocal translocation 3' to the coding region of the gene, w hich

o cc ur re d d ur in g tr an sfe rt i on . T he a ct iv ati ng mut at io n w as f ou nd i n o nl y

1 o f 6 /3-p ro pio lac to ne ind uc ed m ous e sk in tum ors ex am in ed, th e on ly

tum or w ith a transform ing H -ras oncogene. T his is a m uch low er fre

q uen cy o f a ctiv atio n th an th at p rev io usly re ported fo r th e sam e tu mo r

ty pe in du ce d b y p ol yc yc li c a rom at ic h yd ro ca rb on s. T he A Transver

s io n mut at io n i s c on si st en t w it h a p ot en tia ll y d ir ec t m u ta ge ni c e ff ec t o f a

spec if ic 0 -p rop io lac tone-DNA adduc t.

INTRODUCTION

A ctivated c-ras oncogenes have been identified in a large

variety of human and animal tumors (1, 2). In every case

exam ined thus far, the m olecular m echanism of activation of

these genes has been show n to be a single base substitution at

specific sites. C odons 12 or 61 are m ost com monly involved in

the activation of ras genes, although exam ples of activating

mutations at codon 13 (3) as well as other sites (4) have been

rep orted . T he role o f carcin og en an d tissu e spe cificity in o nco

g ene activa tio n, as w ell as th e rela tio nsh ip s betw een che mical

c arc in og en -DNA in te ra ctio ns a nd th e p re cise muta ge nic le sio ns

fou nd in a ctiv ated o nco gene s, h as bee n ad dressed u sin g exp er

im en tal an im al tu mo r m odels (5-1 4).

E arly e xp erim en ta l e vid en ce su gg este d a sp ec ific c orre la tio n

b etw een carcin og en id en tity, a nd on co gen e activ atio n. N M U3 ,

w hich form s adducts on the O 6 position of guanine, uniform ly

activated H -ras by a 12th codon GAransition in all of the

rat m am mary carcino mas that co ntain ed th e ac tiv ated g en e (1 1,

1 4). D MB A, w hich reacts w ith ex ocy clic nitrog en s o f p urine s,

u nifo rm ly activ ated H -ras b y 6 1st co do n m utatio ns in h isto lo g-

ically identical rat m am mary carcinom as, although a sm aller

proportion of tumors (20% as compared to 90% for NMU

harbored the activated H -ras gene (14). Sim ilarly, D MB A ac

tivated H-ras by the sam e codon 61 A Trans ve rs io n, when

used w ith phorbol ester in the m ouse two-stage (initiation/

prom otion) protocol (9) or as a com plete carcinogen (6). W e

have a ls o f ound evi denc e fo r d iff er en tia l a ctiv atio n o f oncogenes

induced by three direct-acting alkylating agents in rat nasal

c arc in om as (7 ).4 N ew e vid en ce in dic ate s th at o nc og en e a ctiv a-

Rece iv ed 5 /1 1/ 87 ; r ev is ed 10/ 22 /8 7; a cc ept ed 10/ 29 /8 7.

T he c osts o f p ub lic atio n o f th is a rtic le w ere d efra ye d 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 tio n 1 73 4 s ol ely to i nd ic ate t his f ac t.

' Supported by Grants CA36342, ES03563, and ES03847 and in part by

C en te r G ra nts C A1 33 43 a nd E S0 02 60 fr om th e N IH .

1 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 , a t N YU M ed ic al C en te r,

In stitu te o f E nv iro nm en ta l M ed ic in e, 5 50 F ir st A ve nu e, N ew Y or k, N Y 1 00 16 .

3T h e a bb re vi at io ns u se d a re : NMU , N it ro somet hy lu re a; DMBA, d im e th yl be n-

zanth racene; BPL, /3-p rop io lac tone; SSC, s tandard sa line-c it ra te .

4 A . E . H oc hw alt a nd S . J. G arte , u np ub lis he d d ata .

tion in animal tum ors induced by chem ical carcinogens m ay

not com pletely correlate w ith carcinogenic etiology. For ex

amp le , b oth K -ra .v a nd N -ra .y a re a ctiv ate d in mou se th ymomas

by either ionizing radiation or N MU5 and in rat renal m esen-

c hymal t umo rs i nduc ed by me thyl (mc thoxyme thy 1 )n itr os am in e

(12).

B PL is a w ell characterized carcinogen that induces tum ors

at th e site o f ex posu re in sev eral rod en t sy stem s (1 5). B ase p air

su bstitu tio ns a nd c hromo soma l a be rra tio ns a re in du ce d b y BPL

in m utagenicity assays (16, 17). W hen incubated w ith D NA in

vitro BPL is highly reactive with nitrogenous bases 1 8 a nd

form s adducts predom inantly at the N -7 position of guanine,

as well as the N-l position of adenine (19, 20), and the N-3 of

cy to sine an d th ym in e (18 ). O ur lab oratory h as p rev io usly iden

tified an activated H-ras oncogene in a BPL induced mouse

skin squam ous cell carcinom a (7). W e now report the m echa

nism of activation of this m ouse H-ras oncogene to be an A

T transversion at the second nucleotide of the 61st codon.

M TER I L S N METHO S

D NA M ediate d G en e T ran sfer. D NA fro m tra nsfec ta nt ce ll lin es w as

ex trac te d u sin g th e s ta nda rd p hen ol-c hlo ro form m eth od , e th ano l p re

cipitated, and resuspended in 10 H IM T ris-1 H IM E DT A, pH 8.O . T he

D NA s w ere digested w ith restriction endonucleases (N ew E ngland

B io la bs ) a cc or din g t o m an uf ac tu re r's s pe cif ic ati on s, r ee xtr ac te d, s us

pended in 10 m M T ris-1 m M E DT A, pH 8.0, and transfected using the

D NA m ediated gene transfer assay as described by W igler et al. (21).

D ig este d D NA (4 0 ig)w as tra ns fec te d as a calc iu m p ho sp hate co pre -

cip ita te in to 25 -c m2 tissu e cu ltu re fla sk s s ee de d the d ay b efo re w ith 5

x 1 0s N IH 3T 3 c ells. T wo da ys fo llo win g tran sfec tio n, c ells w ere sp lit

1 :3 , an d flas ks w ere ra ndo miz ed fo r d oub le b lin d sco rin g. F las ks w ere

f ed twic e w ee kl y, a nd f oc i s co re d a ft er 3 w ee ks .

S ou the rn B lo t H yb rid iza tion s. S ou th ern b lot h yb rid iz ation s w ere

ca rrie d ou t a s d es cribe d by S ou th ern (2 2) an d M an ia tis e t al. (2 3). H ig h

m olec ular w eig ht p rim ary tra ns fe ctan t an d tu mo r D NA s (IS M B )w ere

d ig es te d w ith res trictio n e ndo nu cle ase s (N ew E ng la nd B iolab s), an d

electrophoresed in 0.8% agarose gels, for 22 h at 20 V , after w hich

DNA was t ra ns fe rre d t o n ii ro ce ll ulo se f ilt er s ( 22 ). F ilt ers w er e p re hy -

bridized for 3 h at 65 C n a 6 x SSC solution containing 10 m g heat

denatured sheared salm on sperm D NA , 1% glycine, 50 m M sodium

p ho sp ha te , a nd 2 0% Den ha rd t's s olu tio n ( 0.2% e ac h: p ol yv in yl p yr ol i-

dine, bovine serum album in, and Ficoll). The filter w as incubated

overnight at 65 Cin a 6 x SSC solution containing 80 m M sodium

phosphate, 10% dextran sulfate, 2 m g heat denatured salm on sperm

D NA , a nd 3 x 1 0* c pm /M g h ea t de natu re d nick tra ns la te d p ro be . F in al

s tr in ge nc y w as he s w ere 0 .5 x SSC- 0.1 % s od ium d od ec yl s ulf ate a t 6 5 C

for 30 m in. Filters w ere air dried and exposed to K odak X AR film in

t he p re se nc e o f i nt en sif ie r s cr ee ns a t 70C.

O l ig onuc le ot id e Hybr id iz at io n. O li go nu cl eo ti de h yb ri di za ti on s we re

p erfo rm ed a s d es crib ed b y Z arb l et a i. (1 4). T ran sfec tan t D NA (3 0 M g)

w as d ig es te d w ith D am il i, o r /- //'/ /I IIa cc or di ng t o m an ufa ct ur er 's

s pe ci fi ca ti on s. E le ct ro phor es is wa s c ar ri ed out i n f il te re d 0 .7% aga ro se

g els. T he g els w ere d rie d un de r v acu um at 8 0 Cu ntil co mp letely d ry

( 3- 5 h ). G el s w er e p re hy br id iz ed f or l h a t 5 1 Cn h yb ri di za tio n b uf fe r

(0.9 M NaCl, 0.18 M Tris (pH 8.0), 6 HIM E DTA , 20% Denhardt's

solution, 0.5% sodium dodecyl sulfate, 4 M g tR NA , and 500 M g/m l

so nic ated salm on sp erm D NA ). H ybrid iz atio n w as ca rrie d o ut a t 5 1 C

in h yb rid iza tio n b uffer su pp le me nte d w ith 1 % d ex tran su lfa te , l m g/

m l sonicated salm on sperm D NA , and end labeled oligonucleotide

A. Pell icer, personal communicat ion .

556

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r as T IV T IO N IN M O USE R IN OM

pro be (sp ec if ic ac tiv ity , 0 .6 to 1 x IO 9 cp m / g n on ad ecam er p ro be).

O lig on ucleo tid e n onad ec am e rs w ere en d labe le d w ith 50 0 ^ ( 'i | ' l'|-

A T P (N ew England N uclear) and T 4 poly nucleotide k inase (N ew

Eng lan d B i olab s) f or 3 0 m i n. Pro be re ac ti on s w e re t erm i nate d b y ad di ng

E DT A to a f inal concentration of 20 m M . Probes w ere purif ied using

E lu ti p-D column s (S ch le ic he r and Sdd) .A 5 -u l al iq uo t o f e ac h p ro be

wa s count ed t o cal cu lat e s pe ci fi c ac ti v it y .

A f ter p osth yb rid iz atio n w ash es, th e ge l w as e xp os ed to K o dak X A R

f ilm in th e p re se nc e o f in te ns if ie r s cre en s at 7 0 C .

RESULTS

W e hav e prev iously reported that one of tw o m ouse sk in

squam ous cell carcinom as and one of four f ibrosarcom as in

duced by B PL w ere positiv e in the transfection assay (7). O nly

the carcinom a w as found to contain an activ ated H -ras onco

gene.

A new site f or the restriction endonuclease X b al is created

b y a sp ecif ic A Transv ersio n at th e secon d n ucleo tid e o f the

61st codon of the H -ras oncogene. N IH 3T 3 D N A digested w ith

Xbal showed the normal 12 kilobase H ras homologous se

q ue nc e (Fig . 1 , L an e D ). X b al d ig estio n o f p rimary tran sf ec tan t

3081-1 D N A (Fig. 1, L ane A ), y ielded the norm al 12-k ilobase

band, as w ell as tw o new H-ras hom ologous sequences of ~5

and ~7 k ilobases. In order to determ ine the origin of this

m utation, DN A from the original B PL induced m ouse squa

m ous cell carcinom a and the liv er of the tum or bearing anim al

w ere also digested w ith X bal (Fig. 1, L anes B and C). T he tw o

new sequences w ere present in addition to the norm al I I-r .v

f ragm ent in the tum or but not the liv er D NA , dem onstrating

that this transf orm ing point m utation in 3081-1 originated in

the tum or and w as not an artifact of transfection of the tum or

DN A . N one of the other f iv e BPL induced m ouse tum ors

d ig ested w ith X b al d em o nstrated any H -ras h om o lo go us b and s

in ad ditio n to th e n orm al f ragm e nt.

BCD

Kb

12.0

7.0

5.0

Fig . 1 . S ou th ern b lo t an aly sis o f X b al d ig ested D N A . S ou th ern b lo t an aly sis

o f tran sf ectan t D N A s ho win g R FL Ps . T he D N A s ( 15 n g) w ere electro ph ores ed

i n 0 .8 % ag aro se g el an d t ran sf erre d t o n it ro ce llu lo se f il te rs as d es crib ed u nd er

M ate rials an d M e th od s. Fil te rs w e re h yb ri diz ed t o n ic k tran slat ed v -H -ro s

probe (O NCO R). Filters w ere w ashed to a f inal stringency of 0.5 x S SC-0.1%

sodium dodecy l sulf ate at 65 < and ex posed to K odak X A R film . M olecular

w eig hts [in k ilo base s (A f t)| o f b an ds w ere calcu lated f ro m llin d\\\ c ut X - DN A

s tan dard s. D N A s w e re f ro m : L an e A , tran sf ec tan t 3 08 1-1 ; L an e B , o rig in al t um o r

B PL -4 ; L an e C , l iv er f ro m B PL -4 t um o r-b earin g m o us e; L an e D , N IH 3T 3 .

Oli gonu cle otid e Hyb rid iz atio n Ana ly s is . O li gonu cle otid e h y

bridiz ations w ere perform ed to conf irm the results f rom the

Xba l dig es tion ana lys is . Thi s proce dure re lie s on the diffe re nc e

in ability of a perfectly m atched sequence to form a stable

hy brid relativ e to a hy brid w ith a single base pair m ism atch.

N o nad ecam e r p rob es w ere sy nth esiz ed (A p plied B io sy stem s ;

3 80 D N A sy nthe siz er) usin g th e n orm al m o use H -ras seq ue nce

(6), f rom codon 58 to codon 64, as w ell as the predicted A to

T base substitution m utation at the second nucleotide of the

61 st c od on . O lig on uc leo tid e hy brid iz atio ns w ere p erf orm e d

w ith 3081-1 D NA digested w ith the endonuclease H indlll,

because of the ability of this enz ym e to distinguish the trans

f orm i ng an d n ormal al ele sse e b elow ). H y brid iz atio n w ith th e

nonadecam er specif ic for the w ild ty pe sequence (Probe A ,

CA G CA G GT CA A G A A G A GT A ) show ed 5.7-k ilobase hom ol

ogous sequences in both the norm al N IH 3T 3 and 3081-1 lanes

(Fig. 2, L anes A and B ). T he nonadecam er specif ic for an A to

T tran sv ersio n at th e s ec on d n uc le otid e o f th e 6 1s t c od on (Pro be

B , CA GCA GGT CT AGA A GA GT A ) hybridiz ed only to a 7.0-

k ilobase hom ologous band in the 3081-1 lane, w hich corre

sp on ds to th e tran sf orm in g al ele see b elo w ) (Fig . 2 , L ane D ).

S im ilar h ybridiz atio ns w ith B am H l d ig ested 3 08 1-1 D N A , and

N IH3T 3 DN A dem onstrated that this A to T transv ersion

sequence also hy bridiz ed only to a 3.7-k ilobase hom ologous

sequence in the 3081-1 DN A (Fig. 2, L ane F) and show ed no

signal in the N IH 3T 3 lane (Fig. 2, L ane E). T hus, the trans

f orm i ng 7 .0 -k ilo bas e H -ras h omo lo go us al eleo f th e p rimary

transf ectant 3081-1 w as activ ated by an A to T transv ersion at

the second nucleotide of the 61st codon of the m ouse H-ras

oncogene.

R estrictio n S ite Po ly m orph ism s in th e A c tiv e G en e. R estric

tio n f ragm e nt len gth p oly m orp hism s w ere d etected in th e acti

v ated H -ras gene of a prim ary transfectant. For exam ple, a

nov e l 7 .0 -k ilo bas e re st ri ctio n f ragment wa s d ete ct ed in H indlll

d ig este d p rimary tran sf ec tan t 3 08 1-1 DNA . Howe v er, S o uth ern

analy sis of H indlll digested DN A from the original tum or

(B PL 4), as w ell as f rom the liv er of the sam e anim al show ed

th at th e H in dlll re strictio n f rag m en t len gth p oly m orp hism w as

lim i te d to th e p rimary tran sf ec tan t an d p ro bab ly was an artif ac t

o f tran sf ec tio n. Fu rth er restriction m app in g (data n ot sho w n)

of this transfectant DN A dem onstrated that the norm al 5'

H indlll site wa s no t a lte re d a nd tha t a re arra ng eme nt 3 to the

coding sequence of the m ouse H -ros gene had occurred during

the transfect ion.

D N A f rom the B PL induced m ouse squam ous cell carcinom a

p rimary tran sf ec tan t 3 08 1-1 was d ig es te d w i th s ev eral re stric

tio n en don ucleases b ef ore a sec on dary tran sf ectio n. A s sh ow n

in T able 1, Hindlll digestion did not show any signif icant

decrease in transf ection ef ficiency f rom undigested 3081-1

DN A . S ince the 5' H indlll restriction site at codons 5 and 6

Kb

7.0

5.7

Kb

Fig . 2 . O lig on ucleo tid e h yb rid iz atio n. G en om ic D N A (3 0 p g) w as d ig ested

w ith ///'/ /II (L an es A - D), o r B am H \ (L an es E an d F), an d elcctro ph oresed in

0 .7 % ag aro se g el s. T h e g el s w e re d rie d an d h yb rid iz ed to J2 P e n d-l ab ele d o lig o-

nucleotide probes. D NA s w ere f rom : L anes A , C , and E, N 1H 3T 3; B , D , and F,

3 08 1- 1. Pro be s u se d w e re : L a ne s A an d B . Pro be A ( wi ld ty pe ); L an es C -F, Pro be

B (co do n 6 1 A Transvers ion) . Kb, k i lobases.

55 7

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r a s TIV TION IN M OU SE R INO M

T ab le 1 S e co nd ary t ra ns fe ct io n o f re st ri ct io n d ig es te d DNA

Prim ary tran sf ec tan t D N A (3 60 n g) w as d ig es te d w ith re stri cti on e nd on uc le -

ases (N ew E ng lan d B io lab s), e xtracted w ith p he no l-c hlo ro fo rm , an d eth an ol

p recip itated . D ig este d D N A (3 0-4 0 /ig ) w as tran sf ected in to N IH 3T 3 cells b y

c al ci um phos ph at e c op re ci pi tat io n. N i ne re pl ic at e p lat es w e re s tai ne d an d s co re d

d ou ble b li nd f or e ac h g ro up , af te r 3 w ee ks .

R F R N S

DNAnzyme3081-

3081-

3081-

3081-

3081-

NIH3

T24(primary

transf ectant) N one

EcoRl

BamH\

Hindm

Psll

T3 None

NoneEfficiency

foci/ngNA0.095

0.068

0.080

0.100


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1988;48:556-558.Cancer Res

Anne E. Hochwalt, Jerome J. Solomon and Seymour J. Garte

Carcinoma Induced by an Alkylating AgentOncogene Activation in Mouse SquamousrasMechanism of H-

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