3T3 cells does not suppress the transformed phenotype

Gene, 45 (1986) 215-220

215

Elsevier GENE

1690

Expression

mRNA in H-vus transfected NIH/3T3 cells does not suppress the transformed

of anti-sense

phenotype (H-ras

oncogene;

RNA

duplex formation;

SV40 expression

plasmid;

DNA

rearrangements;

recombinant

DNA)

B. Salmons *, B. Gtoner **, R. Friis, D. Muellener and R. Jaggi Ludwig Institute for Cancer Research, Inselspital, 3010 Bern (Switzerland) (Received

February

(Revision

received

(Accepted

Tel. (031)-261241

lOth, 1986) June 26th, 1986)

July lst, 1986)

SUMMARY

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-rus-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-rus was observed. Anti-sense RNA was present in a lo-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense : anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-rus sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.

INTRODUCTION

Cells in culture can be transformed by a number of viral and cellular oncogenes (reviewed by Bishop, 1985). Several mechanisms have been identified which result in the activation of proto-oncogenes to

* Present Medical

address: College

Department of Georgia,

of Cell and Molecular

Biology,

Augusta,

(U.S.A.)

GA

30912

Tel. (404)-828-3271. ** To whom

correspondence

and reprint

requests

should

be

addressed. Abbreviations: hygromycin

bp, base pair(s); B; kb, kilobase

polyacrylamide;

R, resistant;

ds, double

strand(ed);

or 1000 bp; nt, nucleotide(s); ss, single strand(ed);

HyB, PA,

tk, thymidine

kinase.

0378-I 119/86/$03.50

0 1986 Elsevier

Science Publishers

B.V. (Biomedical

oncogenes (Hayward et al., 1981; Reddy et al., 1982; Tabin et al., 1982; Taprowsky et al., 1983; Capon et al., 198313; Taub et al., 1982). If the identification of oncogenes involved in human tumours progresses and clinical applications are sought, strategies designed to inhibit oncogene expression will be of importance. Several recent reports suggest that the suppression of specific gene products can be achieved by expression of anti-sense mRNA (for reviews see Laporte, 1984; Weintraub et al., 1985). We have attempted to suppress the expression of the H-ras oncogene (Shih and Weinberg, 1982) using anti-sense H-ras transcripts. We were not able to reverse the transformed phenotype of cells transfected with this oncogene. Division)

216

EXPERIMENTAL

(a)

Anti-sense

AND

1985), with a plasmid

DISCUSSION

H-rus

transcripts

are

NIH/H-rus cells supertransfected anti-H-rus construct We have constructed

present

conferring

resistance

to the

antibiotic HyB (Bfochlinger and Diggelmann, 1984) into a clonal line of H-ras transformed NIH-3T3

in

with the SV40

cells (NIH/H-~+a$). Individual

HyBR cell clones were

isolated and grown into mass culture. To determine whether the SV40 anti-H-ras construct directs trans-

a plasmid cont~ning

the first

cription

of RNA with the opposite

polarity to H-vas

exon of the c-H-ras gene linked to the SV40 early promoter in an anti-sense orientation (SV40 anti-H-

transcripts (i.e., anti-sense RNA), total cytoplasmic RNA was prepared from four HyBR cell clones. The

vus; Fig, 1D). The SV40 anti-H-ras

RNA was size-separated by agarose gel electrophoresis (McMaster and Carmichael, 1977), trans-

co-transfected

plasmid

(Wigler et al., 1979; Salmons

was et al.,

C 123

4

5 -

origin

-

28s

+

18s

: ?. :

:

1,3 kb

e-O,7

kb

D Eco

Fig. 1. Antisense

Xbal

RI

H-ras transcripts

cytopfasmic

RNA from NIH/H-raf

respectively)

was glyoxylated

a 32P nick-translated

are present

Bamtil

in NIH/H-rus

for 3 min at SO”C, separated

18 S rRNA diagrammatic (nt 1644-1905

with the SV40 anti-H-ras

supertransfected

on a 1Y0 agarose

construct.

and Carmichael,

1977) and hybridized

in vitro from the bacteriophage

transcripts

are indicated

by arrows.

to

SP6 promoter

H-ras sense RNA, the RNA probe used in (C) recognises

and the 0.7-kb SV40 anti-H-ras

5 pg of total

cell clones I, 2, 3 and 7 (lanes 2, 3, 4 and 5,

gel (MeMaster

probe (A). RNA probes (ss) were synthesized

(Melton et al., 1984). The RNA probe used in (B) recognises RNA. The 1.3-kb H-ras transcripts

cells supertransfected

(lane I) and the SV40 anti-H-ras

6.6-kb H-ras-specific

Bgl II

SV40 anti-H-tax

Cross-hybridisation

with 28 and

is seen with all three probes. Autoradiographic exposure was 3 days (A), 1 day (B), and 2 hrs (C). Panel D shows a representation (not to scale) of the SV40 anti-H-ras construct. The 26t-bp SmaI-Xbal fragment of the H-ras gene in Capon

the XbuI and BamHI vector containing efficient expression

et al., 1983a) was converted

sites of pA8lG

(Paabo

the SV40 early promoter and stability

into a BarnHI-Xbal

et al., 1983) resulting as well as the second

of RNA (Namer

and Leder,

fragment

in transcripts intron

1979).

by linker ligation. with anti-sense

and polyadenylation

This fragment

orientation.

pA8lG

signal from the rabbit

was inserted

into

is an expression j?-globin gene for

217

A

B

C

D

123

123

1 2

12345

ferred to a nitrocellulose

‘b

t

3

,

filter and hybridized

to a

32P-labelled, nick-translated H-rus probe. Authentic 1.3-kb H-rus transcripts are detected in the parental NIH/H-rus cells and in the transfected cell clones 1, 3 and 7 (Fig. lA, lanes 1, 2, 4 and 5). This RNA is also detected probe

that

if the same filter is hybridized recognizes

lanes 1,2,4

H-ras

to an ss

transcripts

and 5). Hybridization

(Fig. lB,

with the ds H-rus

probe (Fig. 1A) or an ss anti-sense

specific rus probe

(Fig. 1C) reveals

in the anti-sense

a 0.7-kb

RNA

H-rus transfected cell clones 1, 2 and 7 (lanes 2, 3 and 5). Thus the SV40 anti-H-rus construct is transcribed into a 0.7-kb RNA in three of the four supertransfected cell clones. 147 133

(b) Anti-sense H-ras mRNA is expressed fold higher levels than H-rus mRNA

-b

at 10-20-

123 90

67

H -ras

anti-sense H-ras Fig. 2. RNase protection transcripts

mapping

of H-ras and SV40 anti-H-ras (A) 10 pg of total cytoplasmic

and duplex formation.

RNA from the NIH/H-ras anti-H-ras

parental

supertransfected

cells (lane 1) and the SV40

4 and 5, respectively)

was hybridized

32P-labelled

RNA

promoter

ss H-ras

Non-hybridized analysed

probe

and complementary

and RNase

for 16 h to a from

to the SV40 anti-H-r03

by separation

Protected

the SP6 transcript.

senting the protected

RNA: RNA duplexes

on a 6% PA-8

followed by autoradiography

by SV40 anti-H-ras

ss RNA probe is indicated.

RNA

complementary

transcribed

to the H-ras

were analysed

as described

senting the protected

gel

repreand the

(B) 10 pg of total cyto-

(lane 1) and SV40 anti-H-rus

1 and 7 (lanes 2 and 3) was hybridized

ss anti-H-ras

were

M urea sequencing

for 3 h. The 350-nt fragment

region shared

plasmic RNA from NIH/H-rascells clones

at 45°C

transcribed

(ss) RNA was digested with RNase A (40 pg/ml)

Tl (2 pg/ml).

32P-labelled

1, 2, 3 and 7 (lanes 2, 3,

cell clones

to a 32P-labelled

from the SP6 promoter, transcript.

RNA.

and

RNA duplexes

in (A). The 133-nt fragment

repre-

region shared by H-rns and the 32P-labelled

ss RNA probe is indicated.

The X-ray film was exposed

to the

gel for seven days. (C) and (D) 10 pg of total cytoplasmic

RNA

from NIH/H-ras

(lane 1) and SV40 anti-H-rtrs

(lanes 2 and 3) was either digested plasmic

RNA: RNA duplex structures

at 45’C

and then digested

to SV40 anti-H-rus

duplexes

by both procedures

formed

to isolate cyto-

(C) or hybridized

to a ‘ZP-labelled

transcripts

detected in clones 1,2 and 7 (Fig. 2A, lanes 2, 3 and 5; 350 nt fragment). Expression of the H-rus (sense) transcripts was determined using radioactively labelled ss RNA spanning the first exon of H-rus. H-rus transcripts share 133 nt with the complementary SP6 RNA probe. Parental NIH/H-rus cells, and the clones 1 and 7, express similar levels of H-rus mRNA (Fig. 3B, lanes 1,2 and 3; 133-nt fragment). Similar levels of ~21’“” expression in NIH/H-rus cells and clones 1 and 7 are observed by Western blotting analysis (Table I). The intensity of the signal

clones 1 and 7

with RNase to hybridize

H-ras transcripts rehybridized

with RNase

Correctly initiated SV40 anti-H-rus mRNA was measured using the RNase protection technique as outlined by Melton et al. (1984). Briefly, radioactively labelled RNA complementary to SV40 antiH-rus RNA was transcribed in vitro from the SP6 bacteriophage promoter. The radioactive RNA probe was hybridized to total RNA isolated from the parental NIH/H-rus and the SV40 anti-H-rus supertransfected cell clones. The RNA was then treated with ss-specific RNase. RNA: RNA duplexes protected from digestion were visualized after gel separation by autoradiography. SV40 anti-H-rus RNA shares 350 nt with the complementary SP6 RNA probe. Correctly initiated anti-H-rus transcripts are

for 16 h

all available

(D). RNA: RNA

were then denatured

ss RNA complementary

and to the

H-ras transcript.

RNA:probe

RNA duplexes were then analysed

as described

in (A). The 133.nt fragment

of homology

shared

indicated labelled

representing

by H-ras and SV40 anti-H-ms

by an arrow. HpaII-digested and served as marker

pBR322

fragments

in bp). The X-ray film was exposed

the region

transcripts

is

DNA was end-

(see left margin;

sizes

to the gel for seven days.

218

TABLE I Summary

of biological

Cell

properties

of the transfected

Morphology

a

clone

cell clones

Soft agar

Tumours

growth b

nude mice”

H-ras

in

fragments

(%I

d

sv40

Human’

anti-H-ras

p2 1 ra5

fragment

(kb)

*

(kb) 51

1

T

2

NT

+ _

23

1.4

2.8

+ _

3

T

13

+

6.6

1.4 -

7

T

57

+

6.6, 15

1.4

+

NIH/H-ras

T

52

NT

6.6, 15, 23 -

-

NIH/3T3

+ _

+ _

a The morphology

0

0

of cells grown

in monolayer

culture

is indicated.

T, transformed

+

-

morphology:

NT, non-transformed,

normal

morphology. b 5 x lo4 cells were plated into medium containing 20 days and expressed ’ Tumourigenicity

as a percentage

of the cell clones was determined

2 weeks and are indicated d The presence by Southern e Expression antiserum,

0.35 % agar. The presence

of the total number

by a plus-symbol.

of H-ras-specific

blot analysis

BarnHI

by subcutaneous

Mice without

DNA fragments

and hybridization

to H-ras-

of human p21 was determined

by Western

generously

supplied

tumours

injection

of

1 x

were observed

was determined

lo6 cells into nude mice. Tumours

and the SV40 anti-H-ras-specific nick-translated

blotting

of cell-membrane

by Dr. R. Sweet, followed

colonies

for an eight-week

or SV40-specific analysis

by immunovisualization

from the RNase-resistant fragment indicative of the anti-H-ras transcripts (Fig. 2A, 350~nt fragment) was compared with that from H-ras transcripts (Fig. 2B, 133-nt fragment). Differences in size of the protected fragments and exposure time of the autoradiograms were taken into consideration. A IO-20fold greater level of expression of SV40 anti-H-ras transcripts was detected. (c) A small fraction of sense H-ras transcripts is duplexed with anti-sense

of anchorage-independent

after

of cells plated.

H-ras transcripts

One mechanism by which anti-sense mRNA might inhibit sense RNA expression is by the formation of RNA : RNA duplexes. These ds molecules can inhibit translation (Paterson et al., 1977). The SV40 anti-H-ras RNA shares 133 nt of sequence complementary to H-W mRNA. RNA was isolated from NIH/H-rus cells and the SV40 anti-H-rus supertransfected cell clones 1 and 7, and digested with RNase. If the two RNAs are duplexed intracellularly, a fragment of 133 nt should be protected from the ss-specilic digestion of RNase. RNaseresistant RNA was detected by denaturation of the duplex and hybridization to a radioactively labelled probe covering the complementary region. After a

EcoRI-BglII

appeared

after

period. DNA fragment

were determined

DNA probes. extracts

using an anti-human

with ‘251-labelled

protein

p2 l’““-specific

A.

second RNase digestion, the protected labelled probe fragment indicative of the in vivo duplexed region was visualized by gel separation and autoradiography. A weak 133-nt signal is detected using RNA isolated from cell clones 1 and 7 (Fig. 2C, lanes 2 and 3), indicating that H-rus and SV40 antiH-W transcripts are present in these cell clones in a duplexed form. The same experiment was carried out after in vitro incubation of total RNA under hybridization conditions. This allows the formation of all possible H-rus : SV40 anti-H-rus duplex molecules. These conditions result in an increased 133-nt signal (Fig. 2D, lanes 2 and 3). No signal is detected when RNA from NIH/H-rus cells is tested in this assay (Fig. 2, C and D, lanes 1). These results indicate that, under normal cytoplasmic conditions, only a small fraction of H-ru.s RNA is duplexed with SV40 anti-H-rus transcripts. (d) The transfected rearranged in SV40

H-rus gene is frequently anti-H-rus supertransfected

cells

The parental NIH/H-rus cell line was obtained by transfection of aplasmid containing a 6.6-kb BumHI DNA fragment encoding the entire H-rus gene

219

(Tabin et al., 1982) into NIH/3T3 cells. Digestion of genomic DNA from NIH/H-ras cells with BamHI, followed by separation on a 0.8% agarose gel, transfer to nitrocellulose and hybridization to a nicktranslated H-ras-specific probe, reveals three hybridizing bands of 6.6,15 and 23 kb (Table I). The two larger fragments probably contain H-ras copies which have lost one of the flanking BamHI sites during transfection and integration. The analysis of the three SV40 anti-H-ras supertransfected NIH/Hras clones reveals that these sublines have lost or rearranged some of these H-ras-specific BamHI fragments. SV40 anti-H-ras supertransfected cell clone 1 contains only the 23-kb fragment (Table I). This fragment contains a functional H-ras gene copy since this clone grows in soft agar and forms tumours upon injection into nude mice (Table I). The nontumourigenic clone 2 does not express H-ras mRNA (Fig. 1, A and B, lanes 3). This clone has lost all the parental H-ras-specific fragments and a novel 2.8-kb fragment is detected (Table I). Clone 3 is tumourigenie and has only retained the 6.6-kb fragment of the parental NIH/H-ras cells (Table I). Both the 6.6and 15-kb fragments are retained in the tumourigenic clone 7 (Table I). The presence of the SV40 anti-Hras construct in the supertransfected cell lines can be determined by restriction enzyme digestion with EcoRI + BglII, followed by hybridization to an SV40-specific probe (Fig. 1D). The indicative 1.4-kb EcoRI-BglII fragment is present in the DNA of cell clones 1,2 and 7. It is not present in clone 3 or in the parental NIH/H-ras cells (Table I). The only clone that shows a phenotypic reversion (clone 2) has lost functional copies of the H-ras gene. We have not been able to reverse the transformed phenotype of H-ras-transfected cells by simultaneous expression of H-ras and anti-sense H-ras RNA. We have obtained similar results with a construct in which the SV40 early promoter is linked, in the anti-sense orientation, to the v-mos gene (Van Beveren et al., 198 1) and transfected into cells transformed by the v-mos oncogene (not shown). Recently Kim and Wold (1985) have demonstrated that even a 300-fold excess of anti-sense tk RNA over sense tk RNA reduces, but does not completely abolish, tk enzyme activity. N. Hasan, G. Somasekhar and W. Szybalski (personal communication) also have found that a large excess of both galK and ,?-phage gene-N anti-mRNAs have little effect on the expres-

sion of the corresponding sense genes. A complete suppression of H-ras expression is probably required to reverse the transformed phenotype of NIH/H-ras cells. Sufficiently high levels of anti-sense H-ras RNA cannot be obtained in supertransfected cells even if the anti-sense construct is transcriptionally regulated by the strong SV40 promoter. The extremely high levels of anti-sense RNA necessary for absolute gene suppression will probably limit its applications.

ACKNOWLEDGEMENTS

We thank Dr. S. Kozma (Ludwig Institute, Bern, Switzerland) for NIH/H-ras cells, Dr. R. Sweet (Smith, Kline and French Laboratories, Swedeland, PA, U.S.A.) for anti-human ~21’“” antiserum, and Dr. W. Schaffner (University ofZurich, Switzerland) for plasmid pA8 1G. We are grateful for the excellent technical assistance of Ms. S. Saurer and A. Schlafli. We would also like to thank Dr. W.H. Glinzburg for critical comments on the manuscript, Ms. C. Wiedmer and M.T. DiabatC for editorial assistance and J. Grtinig and P. Wegmliller for art work.

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