Nuclear oncogene amplification or rearrangement is not involved in human colorectal malignancies

Eur J Cannr C/m Onco~, Vol. 24. No. 8, pp. 1321-1328, 1988. Printed m Great Britam

Nuclear Oncogene Amplification or Rearrangement is not Involved in Human Colorectal Malignancies RICCARDO Experimental

DOLCETTI,

VALLI DE RE, ALESSANDRA

Oncology 1, Centro di Rifrimento

Oncologico,

VIEL, MAURO PISTELLO. MANUELA TAVIAN and MAC‘RO BOIOCCHI*

bi’a Pedemontana

3308 1 .4z~inno (Pordenone),

Occidentale,

It&

have examined 44 cases of human colonic and rectal carcinomas for structural

Abstract-We rearrangement

and amplification

hybridization

of c-myc, N-myc,

L-myc,

showed evidence of c-myc amplrfication

the same tumour also showed a rearrangement

c-myb and ~53 oncogenes.

L)Iv~~)~

in only one of the samples tested. In addition,

immediately 3’ to the c-myc locus. No rearrangement

could be found at the c-myc locus in the other 43 cases. Moreover,

our molecular analp~is of S-

myc, L-myc, c-myb and ~53 genes indicated no relevant alteration of the copy number and/or genomic structure of these nuclear oncogenes. Thus, at least in human colorectal malignancies, it is unlikely that nuclear oncogene structural alterations

and/or ampl$cation

plays a major role in

tumour induction or progression.

INTRODUCTION CELLULAR

proto-oncogenes

by their

homology

acutely

transforming

mediated

gene

suggests

to the oncogenic

transfer

or

techniques

which

their effect stimulating

growth

As

structural of

dred

DNA-

[ 1, 21. Evidence proteins

signalling and

part

of this

picture,

may

growth mem-

[7, 9, lo]. have

proto-

oncogene

activation

neoplastic

state remains

structural

genes

are

oncogene

tumours The

or

frequently

cancers

in several

cell lines

proto-oncogene members,

which

similar

structural

myc

colon We

expression frequently

of this gene colonic

properties

in

carried

and

the

and lung of the cin human of c-myc

cell

been

line

with

(Co10 320) but there was or amplification

in a group

carcinomas

of

which

has initially

cancer

of rearrangement

locus

stage

[ 19-201. Amplification

in a human

no evidence

of 29 primary

of

human

[ 191. out

similar

analyses

on c-myc

and

four other nuclear oncogenes (N-myc, L-myc, cmyb, ~53) to evaluate the real importance of struc-

have

tural

human

[5-lo].

family c-myc,

carcinomas

neuroendocrine the

to occur

ovcrcxpression

of the

retrovi-

translocation

seems

genes,

and rctinoblastomas

[ 10, 17, 181. Increased

found

and/or

chromosome

The c-

is the finding

and L-myc

to the clinical

of neuro-

proto-

of nuclear

demonstrated

characterized share

formation

in the pattern

functions.

a

kin-

to be translocated

interesting

ofN-myc

correlated

gene

colonic

of c-one

by gene amplification,

and tumour myc

been

myc

it appears

alterations

in tumour

expression

development

to be elucidated,

Modifications

insertion

been

the

or regulatory

involved

progression.

and

cellular

Particularly

progression

with between

these

[5, 151 and amplified in the HL60 leukaemia cell line [ 161. In addition,

the amplification

are surmised to play a central cell proliferation and differen-

the relationship

may have related

that

this oncogene is amplified and overexpressed in mammary, lung and colon carcinoma ccl1 lines

[3, 41.

Although

not only reflects

but also suggests

has been found

promyelocytic

exert

nuclear

sequences

lymphomas

pathways

mitogens

this gene grouping kinship

myc oncogene

and cytoplasmic proteins to the a proliferative response may bc

oncogene products role in regulating tiation

factors

by

on cells. By such pathways, signals are conveyed through

brane receptors nucleus, where induced.

of the

identified

sequences

retroviruses

components

through

ral

been

that proto-oncogene-encoded

represent

that

Nevertheless,

have

alterations

of such gents

in human

colorectal

malignancies. has three

N-myc features

wellMATERIALS

and L-myc, [ 1 l-l 41.

AND

METHODS

Normal and neoplastic tissues Normal and neoplastic tissues

Accepted 8 March 1988. *Author to whom correspondence and reprint requests should be sent.

24 patients with from 20 patients 1321

were collected

from

adenocarcinoma of the colon and with adcnocarcinoma of the rec-

1322

R. Dolcetti ct al.

turn.

None

of the 44 patients

ment prior to surgery. mucosa

(dissected

were removed

near

Specimens

and were subsequently In cases

was

unavailable,

excision

structure

was performed

restriction

endonucleases,

at -80°C

until

colonic

ana-

mucosa

blood

Lymphocytes through

was

were Ficoll/

gene.

High

appear

molecular

described

previously

restriction

[22]. DNAs

transferred

Screen

was carried

(New

and 2

N-myc,

L-myc,

c-myb,

Ki-ras

were

The

washed

(twice

filters

under

filters

moderately

for 30 min in 1

intensifying

were

then

screens

Probes The following

SSC at

X

hybridized

to v-

X

1% SDS

SSC at 55°C).

autoradiographed

at -70°C

using

for 18-72 h.

enzyme-generated

1 kb EcoRI-BamHI

fragment

DNA

of pJ B327

(L-myc) [14] (a kind gift from Dr. J. Minna), the 1 kb EcoRI fragment of HiHi (v-Ki-ras) [24], the 2.7 kb EcoRI

fragment

of pHM2A

gene

181 digested

with

DNAs

labelled

were

was

not

(c-mos)

signals

[25],

staining myc

with 32P dCTP

of Feinberg

and

DNA probes resolution

by the random

Vogelstein

the

colonic of sample The

probe

other

at intensities

sequences

in control

181 DNA

gene.

to

a IO-fold ampli-

However,

the

c-mos

on chromosome

or rcarrangcd

8 [25]

in sample

181

in the hybridization

and

occurred

was

corresponding

indicating

c-myc

gent

in sample 18 1 As shown in

of sample

that a specific

c-mos

supported

amplification

the

involving

in this tumour.

Owing

to

in were

primer

(Amersham)

WI.

minute

chromosomes

and

homogeneous

regions.

Furthermore,

the

third

probe

Hind111 In

detected

fragment an

rearranged exclude

BglII,

in carcinoma

181.

to locus

presence

with

from

the enzymes cut at different

end

of the c-myc

6.2 kb in size were respectively

gene.

exon-c23-kb-

characterize

of this

mucosa

and PstI,

better

tumour

of Hind111

DNAs

normal

restriction

181 and (sample SacI,

observed

from 182)

XbaI,

distances

Abnormal

the and

PstI

to site

correwere and

from

the 3’

fragments

of 5

with

BglII

and

(Fig. 3).

Thus it was possible to map the beginning of the rearrangement between the conserved XbaI site and the abolished myc locus.

RESULTS

c-myc-related

c-myc the

which

a novel

attempt

polymorphism, digested

method

amplified

the c-myc

of c-

radiolabelled

tumours.

in DNA

an intensity

c-myc

between

clone

after

by

c-myc does not

normal

or HindIII.

close to c-myc

interpretation

of a c-DNA

gel. DNA fragments

of

band corresponding

(Fig. 2b). The difference

fragment

plasmid

labclled

DNA,

of the

and

agarose

the

of c-myc amplification from diluted DNA.

located

sponding

from

to

EcoRI

to obtain

fication gene,

[26]

prepared

human

oncogcne

was revealed

that of the control

(~53)

were

out-

are seen

the exception

in any of these

Fig. 2a, a IO-fold dilution required

of p53-H13

low-melting-point

(with

to single-copy

The degree was estimated

fragment

myb [27], a kind gift from Dr. Pierotti.

results

the c-myc

compared

to the c-myc

the 1.8 kb EcoRI a 1.2 kb PstI

cut the DNA

to be the germ-line

an extensively

to double restriction

locus

or Hind111

a lack of material it was impossible to perform any cytogenetic analysis of sample 181, with respect

fragments were used as hybridization probes: the 1.6 kb Cla I-EcoRI fragment of pHSR-1 (c-myc) [7], the

mucosa,

DNA

stringent

SSC-0.

X

at 55°C and once for 30 min in 1 The

and p53

twice for 5 min in 2 X SSC at room twice for 30 min in 2 X SSC-1%

temperature.

conditions

of

Blots probed

c-mos

tested

c-myc

EcoRI

which

to be rearranged

corresponding

10” cpm/ml

X

of DNA

cells.

Hybridiz-

SDS at 65”C, and twice for 30 min in 0.1 room

and

England

[23].

( 1.8 X 10” cpm/p.g).

a 32P labelled probe were washed temperature,

Plus

gels

out at 37°C for 18 h in a buffer

50% formamidc

with c-myc,

by the Milano,

agarose

method

with

were used

Reprcsentativc

Thus,

However,

as

were digested

in 0.7%

to Gene

isolated

Boehringcr,

by the Southern

containing

was

as recommended

(Amersham,

electrophorescd

Nuclear) ation

DNA

endonuclcases

manufacturers Italy),

weight

genes amount

of the

using

in Fig. 1. In all DNAs

gene fragments. Southern blotting

the

sample 181), the human c-myc probe detected 12.5 kb EcoRI and 11.8 kb Hind111 fragments, which have been shown

nitrogen.

for

to the filters. blotting analysis

side the c-myc

ofamplification.

and c-mos

control

in liquid

surgical

the normal

in liquid

internal

were frozen after

the extent

the Ki-ras

transferred Southern

20 ml of peripheral

and frozen

as an

of resection)

to measure

In addition,

immedi-

taken in the presence of heparin. then separated by sedimentation hypaque

as a reference

pathologist

stored

where

treat-

and normal

the margins

as soon as possible

lysed.

tissues

by the attending

ately after surgery. nitrogen

had undergone

Neoplastic

PstI

site at the 3’ end of the c-

oncogenes, c-myc, N-myc, L-myc, c-myb and P-53 in the DNA of 44 freshly obtained colonic and rectal

With all the restriction enzymes employed the intensities of the normal c-myc fragments were amplified about IO-fold with respect to the abnormal ones.

carcinomas. Peripheral lymphocytes or normal colonic mucosa DNAs of the same patient were used

To investigate whether the c-myc closely related oncogene N-myc was amplified or structurally

We studied

the organization

offive

nuclear

proto-

~Vuclear

Oncogene

Amplzjicacations

1323

12345678910 Kb

C-fTlyC

ki

12.5,

ras 3.0,

-

Eco

1

RI

ITig. 1. Eco RI digests of DiV,4 r from a representative group ofcolorectal carcinomas

(lanes

was rehybridized levels. Relatiae lane 5),

hybridized

normal

tissues

The same blot

and normal colonic muco~a (sample

18’2,

(sample 18 1, lane 6) showed an intense signal

to the 12.5 kb-Em is ampl$ed

181

u’ith a r-m_vc specific probe.

ubith a Ki-ras probe. indicating relative single-gene copy to the Ki-ras

onetumour DNA

corresponding

and f ram corresponding

2,4,6,8,10)

(lanes 1.3,5,7,9)

RI c-m_vcfragment,

in thisfragment.

indicating

that it

kb = kilobaser.

182

Kb

Kb

23 b 12.5

b

11.8

6.2 ?? 6.0, 5.0’

a (I

??

“.

2.5 b

b

2 Fig.2.

(a) Intimation

ofthe degree oft-mvcgene

3

ampliJicntmn in .sample

181, .Southern blotting ofuarious amounts of D.\‘.4 from tumour 181 mzs compared to 10 kg of D‘V‘4 from the corresponding normal tissue. The results indicate that sample 181 has a greater than lo-jbid increase in co& number, relatille to sample 182. (6) Southern blot ana
of the sameJilter differences

wth n human specrjic c-mos probe

in the intensittes of the 2.5 kb fragmentr

equal amountc (10 ~8) of 181 and 182 DNAs. kb= bases.

kilo-

Fig.

3. I.oc&ation

Ten micrograms

of the c-rqx

ofDNA

from

normal colonic muco~a ofthe digested ubith HindIII 5,b). Hybridi@ion rryr related fragments DNAs,

locus rearrangement

(sample 182. lanes 2,4,6)

tame patient

(lanes 1,2), BglII

of blots

in tumour 181.

tumour 181 (lanes 1.3,5) andfrom

the

were

(lanes 3,4) and Pst I (laner

with a thtrd exon-c-myc probe detected nocel

on!v in sample

5 kb in BglII-digested DNAs).

181 (23 kb in HindIII-digested

Dh’.-ls and 6.2 kb in PrtI-

kb = kilobases.

digested

R.

1324

a. Kb

12

Dolcettiet

h u.

/ 34

Kb

b

al.

5678

7.1 b 6.6b 4.3,

P53

c-myb

N-myc 4 Fig.4.Southern correspondiq

blot analy.riJ of the j~~-qc, c-rryb and ~53 loci in Ijj!:A

normal controls (lanes 2,4,6,8,10,12).

with a specibr N-rrycprobe.

(b) Hvbridization

probe.

of Eco RI (lanes 9,lO) and HindIll

(c) Autoradiograms

,f?om colorectal crrrcinomns (Inner

(n) Era RI (I nne~ 1,2) and UglII

on&ri.c offour

Divas

1,3,5,7,9,1 1)

afler .&a RI (lanes 5.6,7,8)

di,Festion uting o humm c-nyb

(I anew 1 1,12) digested I>,V,-lI hvbridi;ed

with u ~53 probe.

kb = kilobases

1

2

3

4

5

6

7

8

and

(Inner 3,1) digested D,V.-l Llirbridized

9 10

Kb

L-myc 5 Fig. 5. Southern blot rrna!ysis qfrepre~errtatize gerromir D,2:4 !,f. mm colorettnl ndenocorcinorna~ digeJtrd u,ith Erco RI cold probed mith the I.-myc gene. The 10 kb and 6.6 kb positions repwent the Eco RI gerromic I.-m_vc,fragments. kb = kilobasa.

Nuclear Oncogene Amplijicalions modified

in colorectal

EcoRI

and

BglII

cancers,

digested

Southern

DNA

blots

were

of

hybridized

with a homologous specific probe. A similar analysis was carried out hybridizing EcoRI digested DNAs with a human with these in the

L-myc

probe.

two probes

relative

intensity

tumour

samples

mucosa

or peripheral

previously

as compared

lymphocytes

of L-myc

fragment

gene

and 6.6 kb), equally examined For

defined

blot

cellular

DNAs

endonuclease

radioactive

molecular

Our

normal

polymor-

results

and clone

of the

revealed

the

fragments

tested

generated

enzymes

revealed

bands samples,

and

15 kb

the

there

in the genomic

for

organization

in tumour In addition,

to probes

of human

the variant

EcoRI

p53

or in gene copies

did not reveal

of blots

survival.

amplifications

terminal

phases

in some seem

and

other

involvement

experimental

structural

nuclear genes

because

pathway

models,

nuclear

onco-

in the induction

of the

transformed phenotype findings [19, 211, our

[35]. Confirming previous data indicate that c-myc

amplification

very

the

occurred

44 carcinomas

locus.

However,

c-myc

copies

tumour

in sample

previous

of rearranged

181 is probably

indicate

that

process,

remains reports

our find-

hyperexpression observed

amplification

or obvious

structural

changes

not significantly susceptibility.

c-myc

locus.

However,

we cannot

rule

point

mutations

detectable DISCUSSION Although

the normal

oncogenes

hypothesis

an important evidence

cancer

precise

products

Thus,

that analysis

in the tools

in providing

by chromosomal

juxtaposition

of c-abl

to 22 in pH’ chromosome-positive in these

[31],

are useful

malignancies.

rearrangements

a better

Moreover,

examples

the [30]

chromosome

chronic molecular recent

9

myelogen-

in neuroblastoma at a high frequency

oncogenes

[35, 361 as well proteins

in colon

reported

[37-391;

tumours

stability. of

may alter or

In our

although

and

occasional

tumours as

genes

of the p21 in

transfection

have other

studies

technique

on

NIH

of Ki-ras activation in these tumours: three out of 12 carcinomas tested (unpublished results). N-myc is described as undergoing gene structure modifications, and/or mRNA hyperexpression in tumours

of neuroendocrine

a remarkable

ras been

findings

N-myc amplification was and exclusively associated

3T3 cell line, we found

we gross

and c-Ki-ras

as overexpression however,

or

and the c-mos proto-

c-Ha-ras

polyps

may

study

amplification

not shown),

the DNA

mRNA activated

transcription

of the c-Ki-ras

of amplified

malignancies. c-myc

markers

show that oncogene amplification may be associated with tumour progression in specific human cancers. Thus, found

mRNA

(data

[40, 411 using

other

c-myc

evidence

oncogenes

lymphoma

from

no

of more

cases of onco-

of

of

c-myc

not

we used, could

these

of colorectal

regulation

found

benefits

translocation,

in Burkitt’s

the

that

in

is that

at the

or insertions,

methods

locus

to gene out

explanation

in the development increase

of these genes

development

and

[ 1, 21.

important

the two best known

the transposition

ous leukaemia

process

c-myc possible

hyperexpression

c-one genes play

may have

patients

activation

myc/Ig and

suggests

diagnostic

prognosis. gene

that activated

the

be related

or small deletions

by the analytical

Another

of proto-

it is a widely

role in the malignant

and of their for

functions

are not yet well understood,

accepted Recent

affect

cellular

cannot

of

in human

colorectal

tumour

its

to be elucidated.

frequently

malignancies,

a

and

[19, 211 and

the elevated

mRNA,

a

3’ to the c-myc

of the amplification importance

to c-

also showed

the small proportion

observed

in one of

addition

immediately

Therefore, the c-myc

only

In

the same

functional

in patients

rarely,

examined.

rearrangement

ings

to colorectal

the

[34]. In

with breast cancers [29] (data not shown). Thus, it appears that this rare allele at the c-mos locus may contribute

onco-

We chose

in regulating

of the mitogenic

to be implicated

the actual

and

of these

supposed

in any case

allele of 5 kb observed

patient

structure

the

of their

relatively

was to determine

malignancies.

consequence

as compared

occurs

colorectal

in human

genomic

samples)

hybridization

of gene

More amplifi-

and that it is associated

and overall

myc amplification,

gross alter-

specimens

c-mos

EcoRI

HindIII-digested

EcoRI-digested

to normal

of

only specific

was no detectable

of the p53 gene ones.

examined

6.8 kb

for

(Fig. 4~). Thus,

and

relapse

that

of c-myc

fact,

no

oncogene

cancer,

related

[lo].

rearrangements

genes

to a s2P labelled

in breast

subtype

genes

6.6 and

analysis

by Hind111

hybridized

of the HER-2/neu

The aim of our study

of three

(Fig. 4b) with Further

in all tumours

(3.0

of 4.3,

cation

to analyse a

c-myb

presence

of gene amplifications.

restriction

human

bands

7.1 kb in all the samples

with

to be

et al. [33] showed

prevalence

with Hind111

hybridized

reported

recently,

Slamon

the three to be ampli-

[ 10, 14, 321 and

carcinomas was

histological

with disease

locus,

lung

that such of tumours

In addition, were found

to an aggressive

(10.0

of the c-myb

were digested

c-myb-hybridizing

cvidencc

ation

length

form.

amplification

frequently

in the 44 patients

analysis

restriction

probe

As

endonuclease

(Fig. 5).

Southern

genomic

DNA

colonic

by two alleles

represented

fied in small-cell

in

(Fig. 4a).

EcoRI

malignant

of the myc family

c-myc

bands

III and IV [17], suggesting is involved in progression

to a more

of blots

of the

with stages an alteration members

any alteration

to normal

[ 141,

a restriction

phism

gene.

or sizes

described

revealed

Hybridization

did not reveal

1325

origins.

incidence

However,

no

R. Dolcetti

1326

information is available about its involvement in colorectal cancers. Since gene amplifications are the almost unique genomic structural alteration described, we analysed this phenomenon in our colorectal carcinoma samples. Our data exclude the possibility that N-myc amplification occurs with a significant frequency in these tumours. Moreover, we found no evidence of major gene rearrangement, although more detailed analyses are needed to confirm this preliminary finding. Since current studies suggest that individual members of the myc family may have important roles within specific cell lineages, we carried out a molecular analysis also at the L-myc locus. Hybridization of Southern blots did not reveal any alteration in the relative intensity of the bands in tumour samples as compared to normal counterparts. In addition, the frequency of the two EcoRI-L-myc alleles in the 44 patients examined were as previously described [ 141. A previous report [42] described amplification and hyperexpression of the c-myb gene in two cell lines independently derived from a single adenocarcinema of the colon. Due to the high structural complexity of the c-myb locus [43], we were able to analyse only amplification of this gene and eventually major rearrangements. Again, our analysis did not reveal any structural modification affecting the c-myb gene in colorectal carcinomas. We do not know whether the amplified c-myb gene in the previously described colonic adenocarcinoma cell lines is a characteristic restricted to a small fraction of the colorectal tumours not represented in our samples. However, since oncogene amplification has mainly been found in cell lines ofvarious origins, it is likely that in some cases such a structural

1.

2. 3. 4. 5.

et al. abnormality could arise during the course of propagation of established cell lines. We also studied the structural organization of the ~53 gene, whose product has been detected in several human cancers [44]. It has been proposed that such a nuclear oncogene-encoded protein may play a role in cell cycle regulation, in the induction of immortality and possibly in the transformation of certain non-human cells [45-471. Skin fibroblasts from patients with adenomatosis of the colon and rectum were shown to express elevated amounts of the p53 antigen [48] and the overexpression of this gene was supposed to be an early event, possibly associated with initiation and promotion of inherited colonic cancer. Our molecular analysis of the p53 gene indicated no relevant alteration in the copy number and/or size of this nuclear oncogene, suggesting the role of oncogene activation mechanisms other than amplification or rearrangement in colon and rectum tumours. Thus, at least in human colorectal carcinomas, nuclear oncogene structural alterations and/or amplifications are unlikely to play a major role in tumour induction or progression. In addition, it appears that nuclear oncogene amplification or rearrangement cannot serve for diagnostic or prognostic purposes in this disease. Acknowledgements-We would like to thank Dr. J. Minna and Dr. M.A. Pierotti for the gifts of L-myc and c-myb probes, respectively. The authors thank Paola Pistello for her expert secretarial assistance and also the C.R.O. Scientific Library. This work was supported by grants from the Consiglio Nazionale delle Ricerche: Progetto Finalizzato ‘Oncologia’ i’io. 86.00634.44 and from the Associazione Italiana per la Ricerca sul Cancro.

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