Demethylation of the type I procollagen genes in transformed fibroblasts treated with 5-azacytidine

Vol. 124, No. 1, 1984 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNiCAT\ONS Pages

15, 1984

DEMETHYLATIDN

OF THE FIBROBLASTS

TYPE I PROCOLLAGEN GENES TREATED WITH 5-AZACYTIDINE

M. Iqbal

and

Wieland

Research Unit UCT Medical 7925, South Africa

Observatory

September

6,

TRANSFORMED

Gevers

MRC/UCT Muscle Medical Biochemistry,

Dept. Received

Parker

IN

236-243

School

1984

lung fibroblast results in inactivation genes. No type I collagen or procollathese transformed cells, as determined by polyacrylamide, gel electrophoresis. Analysis of the methylation patterns of these genes showed the type I procollagen genes to be hypermethylated at certain cytosine residues in the transformed cells. However, several of the cytosine residues were methylated in the normal cells where these genes are expressed. These methylation patterns can be altered by treatment of the cells with 5-aracytidine or 5-azadeoxycytidine, but without a resultant activation of the type I procollagen genes. These results show that demethylation alone is not sufficient for gene activation, but that other signals are also required. o 1984 Academicpress, m. SUMMARY. Transformation line, WI-38, with simian of the type I procollagen gen mRNA is detected in

Several

INTRODUCTION.

review when

see

ref.

compared

cells

11.

replicative

event,

cytidine

tion

in (4)

0006-291X/84

of

loss and

expression.

Copyright All rights

is

position

results

the

cells

or with

of this

In

the

to

very first

and

treated its

profoundly often

studies

236

with

the

by

S-

unstable

derivative, nitrogen

by

the atom

at

situation

This

decreased

5-azacytidine

the

a post-

the

(3).

accompanied

is donated

deoxy

ring

$1.50

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

being

to

in

methylation

methylate

pyrimidine

enzyme is

the

unable

modified

occurring

group are

eukaryotic

are

90%

methyl

(for

hypermethylated

In normal

cytosine

5-atacytidine

DNA methyltransferase

are

cytosines

about

of

expression

gene

genes

the

This

When

analogue

5'

of

CpG.

adenosyl-methionine.

of

methylation

counterparts.

with

with

implicated

inactive

3-5%

(21,

embryonic

regulation

active

sequence

human

40 (SV40)

have

Usually

approximately

5-methylcytosine

the

the

to their

dinucleotide

the

studies

in

residues

cytosine

of

virus

methyla-

DNA

altered was

shown

gene to

Vol. 124, No. 1, 1984

induce as

conversion

well

also be

BIOCHEMICAL

as

to

shown

WI-38

human

type

I

inactive

genes

5-azacytidine

previously

collagen,

this

study

SVWI-38 that

we

cells no

gross

type

can

be

as those

such

and

to the

is

type

in

by

produced.

the

DNA, the

a2

normal

demethylated

observed

(5,6)

studies

retroviruses

of

(SVWI-38)

al(I)

that

of

or

a line

that

its

I collagen

demethylation

(7,8,9)

compared

show

myotubes

Several

fibroblasts

that

when

to

have

(10)

can

treatment.

lung

and

fibroblasts

adipocytes.

shown

embryonic

hypermethylated

In

C3H/lOTi and

by

have

mouse

chondrocytes

that

activated We

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

case

produce

not

are

genes

fibroblasts

(11).

genes

in

the

treatment,

5-azacytidine

mouse

transformed

procollagen

procollagen

phenotypic of

does

WI-38

Also, no

SV40

in

spite

of

conversions C3H/lOTi

but

inducing

occur

fibroblasts.

METHODS Cell Culture. WI-38 (ATCC cL75) and SVWI-38 cells were cultured inEagle’s basal medium containing 10% heat-inactivated fetal calf serum. Treatment with 5-azacytidine (AzaC) or 5-azadeoxycytidine (AzaCdR) was either for 18 hours during log phase, or for 8 hours during the S phase, after synchronization with

a double

thymidine

~~$~;;;li;~n;hesis. Amersham) and 50 ug/ml and centrifuged debris. The to acetic acid

ascorbate removed cellular respect

hours

at

samples lamide

15oC. After were lyophilized

gels

block

(20).

Cells were labelled with 10 $X/ml for 24 hours in the oresence of 50 us/ml $-aminopropionitrile. The mediui -was at 10000 x g for 10 minutes to remove supernatant was adjusted to 0.5 M with and digested with 100 ug/ml pepsin for 16 neutralization and extensive dialysis, the and analyzed on 20 cm long 7% polyacry-

(12).

Cells from 150 cm2 flasks were M NaCl, 0.015 M Na citrate), 1% SDS digested with 100 pg/ml proteinase K (Merck) for 1 hour at were then deproteinized by repeated phenol 5ooc. The samples 7 After digestion with extractions as previously described (11). the indicated restriction endonucleases (New England Biolabs, USA, or Boehringer), the DNA was electrophoresed on 1% horizontal agarose gels and transferred to nitrocellulose as described by Southern (13). DNA was digested with 2 units of enzyme/ug ONA for 2 hours at 370C. Completeness of digestion was monitored by The nitrocellulose addition of A-DNA to some of the samples. chicken type I procollagen sheets were hybridized to 32P-labelled clones (14,15) pCg 45 (a2) and pCg 54 (al(I)), as previously described (11). These clones were generously supplied by Dr. All recombinant DNA Helga Boedtker (Harvard University, USA). experiments were done in compliance with NIH guidelines.

DNA Eed and

isolation

in

and hybridization. 2 x Ssc(SSC - 0.15

237

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 1, 1984

analysis. Cells were grown to confluence in F rice of 50 pg/ml ascorbate. Three days The prior to harvesting, l5-J -proline was added at 1 pCi/ml. left behind on cells were lysed with 0.05% SDS, and the matrix the dish was washed 3 times with phosphate-buffered saline and 3 times with 96% ethanol. After drying, the matrix was digested with 100 pg/ml trypsin in 10 mM Tris pH 7.6, 10 mM CaCl, for 20 hours at 370C. The digested material was removed for scintillation counting and the residual matrix digested with 100 pg/ml collagenase (Worthington CSLPA) in the above buffer containing 2.5 mM n-ethylmaleimide in order to inhibit non-specific Extracellular 30 mm dishes

proteases

matrix i-the

(141.

RESULTS

of

Analysis

SVWI-38 duce

fibroblasts

only

matrix,

in

total

but

the

much

more

the

1).

The

SVWI-38

radioactivity decrease

than

aza

C or

amounts

of

Analysis

of

would

aza

CdR,

no

be

on

--Table

into

Total

Collagenase dpm 16565

49242

i 1222

SvwI-38

16854

t

svwI-3a azaC

la235

svwx-38 aza CdR

la413

a higher

treatment were

the

sensitive

2 238

extracellular gels

further

matrix SVWI-38

Extracellular

% Collagen in ECM 34

1474

f 96

a.7

f 618

1631

f 121

a.9

2

1473

-f

742

512

238

115

of

observed

-1

dpm in ECM

WI-38

to

polyacrylamide

collagen deposition in the extracellular and SVWI-38 fibroblasts as well as fibroblasts after treatment with aza C or aza CdR. matrices were prepared as described in Methods. line

to measure

used

labelled

Measurement of (ECM) of WI-38

Cell

overall

radioactivity

changes

deposited

normal

the

extracellular

After

significant

and pro-

an

the

was

proteins.

collagens

by

sensitive

L3d-proline

other

fibroblasts

into

would

WI-38

displayed

collagenase

collagen the

cells

deposited

in

by

produced

collagen

collagens

activity

matrix.

of

produced

transformed

the

the

specific

the

that

Since

components,

with

matrix

pronounced.

matrix

in

25%

(Table

decrease

cells

showed

about

fibroblasts

was

extracellular

the

Vol. 124, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1. Collagen synthesis in WI-38 and SVWI-38 cells. Collawere labelled and isolated as described in Methods. The arrows indicate the position of migration of the al(I) and a2(I) collagen chains. Samples are collagens secreted by primary human embryonic lung fibroblasts (lane l), SVWI-38 cells (lanes 2 and SVWI-36 cells treated with aza C (lanes 4 and 5), SVWI-38 31, treated with aza CdR (lanes 6 and 7) and WI-38 cells (lanes 8 and 9). Odd numbered lanes are samples treated with Bmercaptoethanol. Fioure gens

indicated

that

any

of

the

aza

C or

aza

CdR

synthesized.

major

collagen

type

are and

produced

by

in

counterparts

absent

the

(Figure

after

digestion

of in

clear

from

Figure

1 is

SVWI-38

was that

revealed

SVWI-38

2).

with

In

the between

transformed

239

and

under

cells

with

collagen

the

types

fact

that

the

bridged, amount

no

Analysis

compared

or Mspl,

Hpall

the

that

these

0.5

the

significant

(11).

when

1)

disulphide

shown

that

cells

not a

previously

fibroblasts

fragments

endonuclease

treatment changes

possibility

genes

(Figure

in

by

SVWI-38

the

thus

produced

result

We have

(x2(1)

the

not Also

collagen.

methylated

tion

did

out

was

tested;

produced

ruling III

I collagen

type

conditions

being

thus

no

I mRNAs

type of

was

the

al(I)

genes

were

hyper-

with

their

normal

cells

the

2 kb indicating

were

restriccompletely

methylation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 1, 1984

Fiaure normal indicated

Methylation transformed

2. and

status

the al(I) and a2[I) genes Samples were digested with endonucleases and hybridized to either the a2(1) probe (6). Arrows indicate of i Hind III fragments.

restriction probe (A) or position of migration al(I)

of

both

neither

cytosines

in

these

isoschizomers

of

methylated.

These

methylated

even

Treatment gross

clones

procollagen

gels used

in

the the the

showed

were

if

aza

aza

as

judged

DNA

covered

observed

demethylation

was occurring

over

the

of

what

concentrations

of

the

incorporated

more

efficiently

higher

were

found

deoxy

analogue into

to

be

toxic

240

the

of

the

since

than cells.

aza

in

ethidium

even

entire

used

DNA to

from

though

the

would

were

cells).

CdR resulted

3'-end

the

were

WI-38

Therefore, the

are

cytosines in

C or

since

cytosines

some

(i.e.

shown).

study

both

that

active

total

not

sequences,

cleave

with

(data

representative

doses

also

the

this

genes,

will

genes

of

recognition

CCGG

cells

demethylation

bromide-stained the

the

the

of

the

results

when

in

of

cells.

have gene. it

I

type been Lower would

C and Analysis

be also, of

Vol. 124, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cells were treated with either 5-aza C (30 PM) SVWI-38 CdR (3 @l), the DNA isolated, and restricted with the Samples were hybridized to either the indicated endonucleases. the Arrows indicate al(I) probe (A) or the a2(1) probe (6). Figure

3.

or 5-aza position

the

I

type

treatment

several

cytosines

cells

expressing

play

a significant

sites

would

lysis

since

cleave

them.

genes

(Figures

cells both

of X Hind III

procollagen

revealed

normal that

of migration

bands

2 and in

type

some

have

neither

in

been

the

of

cells

3).

these

CCGG

gene

cells

addition

to

those

genes,

were and

normal

produce

type

fact

methylated

in did

not

methylated

Hpall/Mspl

have

genes

in

the

These

would these

observed

presumably

regulation. the

analogue

confirms

sequences

enzymes

to

after

finding

in

although failed

SVWI-38

This

detected

Nevertheless,

demethylated,

in

I procollagen role

not

in

fragments.

been

were

anaable

to

extensively

I collagen.

DISCUSSION

transformation

Malignant accompanied

by

several

changes

of in 241

eukaryotic phenotypic

cells expression.

is

usually One

of

Vol. 124, No. 1, 1984

the

major

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

changes

extracellular

observed

matrix

fibronectin

(17)

synthesis

of

diminished

and

during

determine

type

I procollagen

dine

analogues

vate

the

normally

(81,

and

in

aza

patients

demethylated

However,

in

treatment,

observed

in

cells

the

case

results

show

methylated

even

tion

a role

plays

certain other

sites

genes

in

DNA

SVWI-38 undergo

mouse

C3H/lOTi

as

will

the

are

not

respond

in

aza C or aza conversion

phenotypic

fibroblasts

as

extensively

after

the

baboons

aza CdR may be

became

cells

cytiacti-

well they

cells

all

of

to

anemic

C or

aza

the

used

where

line

not

not

since

I

that

some

the

cytosines

tissues,

regulation

crucial.

must

mechanism

type

in

also no

and

other

some

in

is

the

were

was

if

fact with

methylathen

only

that

some

methylation

synthesized

extensively

are

genes

that

conjunction

collagen

genes

active

expression,

gene

evident

operate I

in implying

of

Also

type

procollagen

in

cells

demethylated

aza C or aza CdR treatment.

ACKNOWLEDGEMENTS. South

and

expressing

in

are

cytosines,

whose

after

of

shut-off

(3). Our

of

did

been

our

affected

of the

C has

with

though

WI-38

are

this

(91,

cell

that

Even

they

Aza

treatment so

these

reverse

globin

a rat

with

In

genes

8-thalassaemia

in

associated

by administration

CdR.

with

manner.

is

decreased

interaction.

to

fetal

species-specific

identical

CdR

aza

dormant

(19). and

an

C and

genes

expressed

attempted

expression

gene

metallothionein

tissue

we

Such

procollagen

the

(11,15,16),

(18).

components

several

of

collagen

cell-substratum

how

transformation,

as

acid

tissue

and

expression

such

hyaluronic

connective

to

a decreased

components

cell-cell

studies

is

African

This

Medical

work Research

was supported Council

242

by and

the

grants National

from

the

Cancer

Vol. 124, No. 1, 1984

Association.

BIOCHEMICAL

We

for

Boedtker

wish

providing

to

express

the

type

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

our

appreciation

I procollagen

to

Dr.

Helga

clones.

--REFERENCES

1.

Riggs,

40, 2. 3. 4. 5. 6i 7. 8.

A.D.,

Naveh-Many,

T.,

2352-2354. De

Heller, Acad. Sci. De Simone,

Ley, T.J., Humphries,

R.K.,

11.

292, 311-317. Parker, M.I.,

16. 17. 18. 19. 20.

Cedar,

and

J.,

Simone, Natl.

Nienhuis, Groudine,

15.

(1983)

in

Advances

Cancer

Res.

H.

Proc.

(1981)

Natl.

Acad.

Sci.

and Taylor, S.M. (1980) Cell 20, 85-93. and Jones, P.A. (1979) Cell 17, 771-779. Constantinides, P.G., Jones, P.A., and Gevers, W. (1977) Nature 267, 364-366. Constantinides, P.G., Taylor, S.M., and Jones, P.A. (1978) Dev. Biol. 66, 57-71. Venolia, L., Gartler, S.M., Wassman, E.R., Yen, P., Mohanand Shapiro, das, T., L.J. (1982) Proc. Natl. Adad. Sci. 79,

10.

12. 13. 14.

P.A.

78, 4246-4250. Jones, P.A., Taylor, S.M.,

Proc. 9.

Jones,

and

l-30

Res. 10. Laemmli;

P., 79,

Turner, (1982)

A.W. M., Eisenman,

Judge,

5877-5891. U.K ; M(197D)

Hall, L., 4428-4431. J., Anagnou, D.H., Young,

New

Eng.

R.,

and

K., Nature

227,

Zwiers,

N.P., N .S.,

J. Med. Weintraub,

Gevers,

and

and

W.

(1982)

D.

(1982)

Heller, G.H., Heller, P., and 307, 1469-1475. H. (1981) Nature Nucleic

Acids

680-685.

Southern, (1975) .I. Mol. Biol. 98, 503-517. Peterkofsky, ‘e :, and Diegelmann, R. (1971) Biochemistry 10, 988-994. Parker, M.I., and Fitschen, W. (1980) Nucleic Acids Res. 12, 2823-2833. Sandmeyer, S., Smith, R., Kiehn, D., and Bornstein, P. (1981) Cancer Res. 41, 830-838. Klagsburg, M. (1976) Biochim. Biophys. Acta 451, 170-183. Temin, H.M. (1956) J. Natl. Cancer Inst. 35, 679-693. Compere, S.J., and Palmitter, R.D. (1981) Cell 25, 233-240. Bootsma,

33,

D.,

Budke,

L.,

and

Boss,

301-309.

243

0.

(1964)

Exptl.

Cell

Res.