DNA methylation in normal and SV40-transformed human fibroblasts

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1379~1384

Voi. I02, No.4,1981 October30,1981

DNA M E T H Y L A T I O N Edward

IN N O R M A L

S. Diala,

AND S V 4 0 - T R A N S F O R M E D

HUMAN

M a t t h e w M. Plent, Dennis Robert M. Hoffman.

W.

FIBROBLASTS.

Coalson

and

D e p a r t m e n t of Pediatrics, M-009, U n i v e r s i t y of C a l i f o r n i a Diego School of M e d i c i n e , La Jolla, CA. 92093.

at San

Received September 14,1981

SUMMARY The 5 - m e t h y l c y t o s i n e base c o n t e n t of DNA in both normal and S V 4 0 - t r a n s f o r m e d h u m a n d i p l o i d f i b r o b l a s t cultures was m e a s u r e d by high p e r f o r m a n c e liquid chromatography. In c o n t r a s t to other r e p o r t e d studies c o m p a r i n g normal and o n c o g e n i c a l l y t r a n s f o r m e d cells, no a p p a r e n t d i f f e r e n c e was o b s e r v e d in the 5 - m e t h y l c y t o s i n e to c y t o s i n e base ratios in the two cell types. The a d v a n t a g e s of this m e t h o d are also discussed.

INTRODUCTION Questions as to w h e t h e r

have

been raised

there

is a c o n n e c t i o n

and DNA m e t h y l a t i o n is.

To the

indicate

latter

other during

instances oncogenic

SV40-transformed there

and,

methylation MATERIALS

there

an increase

is i n v o l v e d

no a p p a r e n t

as m e a s u r e d

oncogenic

the nature

transformation

of e v i d e n c e

or a d e c r e a s e

human

in their

We report

by high p e r f o r m a n c e

(3,5,9)

in four

lines

genomic

liquid

In

occurred

that

fibroblast total

which

transformation.

in DNA m e t h y l a t i o n

(6,10).

(l-10)

of that r e l a t i o n s h i p

are two lines

(1,2,7,8,11)

and in four normal difference

of i n v e s t i g a t o r s

with oncogenic

change

transformation

is no a p p a r e n t

between

if so, w h a t

question,

that e i t h e r

in DNA m e t h y l a t i o n

by a n u m b e r

studied

DNA

chromatography.

& METHODS

Cell Lines - The normal d i p l o i d human f i b r o b l a s t cells used in this study were MGF287 and MGF292 w h i c h were from p u n c h b i o p s i e s i s o l a t e d at the M a s s a c h u s e t t s General Hospital, Boston, MA., the strain WI38 from human e m b r y o n i c tissue, o b t a i n e d from the A m e r i c a n Type Culture C o l l e c t i o n Cell R e p o s i t o r y (designation CCL75), and BA, a human f o r e s k i n culture from the l a b o r a t o r y of

0006-291X/81/201379-06501.00/0 1379

Copyright © 1981 by Academic Press, Inc. A II rights o f reproduction in any form reserved.

VOI. 102, No. 4, 1981

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Prof. J.A. S c h n e i d e r , U n i v e r s i t y of C a l i f o r n i a at S a n D i e g o S c h o o l of M e d i c i n e . The SV40-transformed h u m a n c e l l s w e r e PI, a s u b c l o n e o f W I S V A 2 (12), P5, a s u b c l o n e of S V 8 0 (12), W I 3 8 V A I 3 s u b l i n e 2 R A (SV40-transformed WI38, ATCC #CCL75.1) and GM0637, from The Human Genetic Mutant Cell Repository, I n s t i t u t e for M e d i c a l R e s e a r c h , C a m d e n , N.J.

NORMAL

S V 4 0 - TRANSFORMED

A

A

C

I00

G

C T

G

~50_

m ~

m 5 Cyt

I I I 246

I 9

I 14

I

I I

246

I

I

9

14

Cyt

time ( m i n ) Chromatograms

of DNA Bases from Normal an~ SV40-transformed

Human Fibroblasts. DNA was prepared from cell cultures and analyzed by high pressure liquid chromatography, as indicated in the text. 20 to 40 micrograms (pg) of hydrolyzed DNA were analyzed in each run. The flow rate of the elution buffer was 2.5 ml per minute at a pressure of 1500 pounds per square inch. The chromatogram of the normal cell line is WI38 while that of the SV40-transformed line is WI38VAI3. All samples were run at an initial sensitivity scale of 0.08 and subsequently increased to 0.01 sensitivity scale after 8 minutes to ensure detection of 5-methylcytosine. T represents Thymine, G, Guanine, C, Cytosine, A, Adenine and m5Cyt, • 5-methylcytosine. UV detection was at 280 nanometres.

1380

Vol. I02, No. 4, 1981

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cell Culture - Cells w e r e grown in m o n o l a y e r s in C o m i n g 490 cm 2 roller b o t t l e s c o n t a i n i n g 75 mls of m o d i f i e d E a g l e ' s M E M (12) s u p p l e m e n t e d w i t h 10% fetal calf serum and p e n i c i l l i n / s t r e p t o m y c i n (50 u n i t s / m l and 50 pg/ml, r e s p e c t i v e l y ) . Cell m o n o l a y e r s w e r e d e t a c h e d w i t h 0.25% trypsin, w a s h e d w i t h p h o s p h a t e b u f f e r e d saline, c o u n t e d in a C o u l t e r - c o u n t e r and finally p e l l e t t e d by centrif u g a t i o n at 4C. DNA I s o l a t i o n - DNA was e x t r a c t e d from i s o l a t e d whole nuclei by s t a n d a r d t e c h n i q u e s (13), t r e a t e d w i t h r i b o n u c l e a s e A, e x t r a c t e d w i t h a s o l u t i o n of p h e n o l / c h l o r o f o r m / i s o p e n t y l alcohol (ratios were 25:24:1), and p r e c i p i t a t e d w i t h 95% ethanol. The final DNA p r o d u c t was l y o p h i l i z e d and then h y d r o l y z e d to bases by res u s p e n d i n g in 200 p l of 88% formic acid, sealing the m i x t u r e in a glass ampoule and h e a t i n g at 180C for 20 min. (i0). The h y d r o l y s a t e was e v a p o r a t e d to d r y n e s s u n d e r n i t r o g e n and r e s u s p e n d e d in 200 ~ i of 100 m M HCI. In control e x p e r i m e n t s w i t h c y t o s i n e (C) and 5 - m e t h y l c y t o s i n e (m5Cyt), w h i c h is the only m a j o r m e t h y l a t e d base found in v e r t e b r a t e DNA, we have found that this m e t h o d of acid h y d r o l y s i s does not affect these bases. This is in a g r e e m e n t w i t h the m e t h o d of Riggs (i0) but not w i t h the results of Ford (14). Uracil was not d e t e c t e d in our p r e p a r a t i o n s i n d i c a t i n g that any RNA c o n t a m i n a t i o n was b e l o w i n t e r f e r i n g levels. A n a l y s i s by H i g h P e r f o r m a n c e Liquid C h r o m a t o g r a p h y (HPLC) The bases r e l e a s e d by formic acid h y d r o l y s i s w e r e a n a l y z e d by HPLC. Samples (20-50 pl) were eluted t h r o u g h an A l t e x U l t r a s i l - 1 0 C X column (0.46 x 25 cm) w i t h 0.02M a m m o n i u m phosphate, pH 2.3, at a m b i e n t t e m p e r a t u r e w i t h d e t e c t i o n at 280 nm, using a m e t h o d m o d i f i e d from Riggs (I0). The data were p r o c e s s e d by a S h i m a d z u C - R I A c h r o m a t o p a c i n t e g r a t o r and c a l c u l a t e d using standard curves. C a l c u l a t i o n s - The p e r c e n t m e t h y l a t i o n was c a l c u l a t e d by the ratio of n a n o m o l e s (nM) m 5 C y t to nM m 5 C y t plus nM c y t o s i n e m u l t i p l i e d by i00. C a l c u l a t i o n s of the n a n o m o l e c o n c e n t r a t i o n s of m 5 C y t and of c y t o s i n e were d e t e r m i n e d by linear r e g r e s s i o n a n a l y s i s using values supplied by s t a n d a r d curves.

RESULTS

and D I S C U S S I O N

Figure pressure

1 shows

liquid

chromatography

and t r a n s f o r m e d readily

cells.

The c a l c u l a t e d

to 3.18,

obtained

of h y d r o l y z e d

from high

DNA from normal

As can be seen in Fig.

while

from 2.90

cell

transformed

data are

shown

for the four normal

determinations normal

patterns

i, m 5 C y t

is

detected.

methylation

was

the e l u t i o n

the range to 3.03. is 2.94

for the

types

and 3.00

lines.

Thus,

lines

i.

The p e r c e n t

ranged

from 2.83

four S V 4 0 - t r a n s f o r m e d

The m e a n + 0.28

in Table

cell

for the total

number

(51 determinations)

+ 0.28 at m o s t

1381

is slight

of HPLC

for the

(53 determinations) there

cells

for the

but p r o b a b l y

Vol. I02, No. 4, 1 9 8 1

TABLE i.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Extent of DNA Methylation in Normal and SV40-transformed Human Fibroblasts.

Cell Line

% methylation ± stand.dev.

# of determinations

A. Normal MGF 287 MGF 292 WI38 BA

2.83 + 0.25 3.04 0.17 2.87 0.36 3.18 0.23

20 12 ii 8 51 total 2.94 + 0.28

Total mean B.

SV40transformed

P1 P5 WI38VAI3 GM0637

3.01 + 0.32 3.03 0.30 2.90 0.24 3.01 0.19

21 15 8 9 53 total 3.00 + 0.28

Total mean

Cell cultures were grown in 490 cm 2 Corning roller bottles containing 50 - i00 mls of Eagle's MEM with 10% dialyzed fetal calf serum. The cells were harvested by trypsinization, washed twice with phosphate buffered saline and the DNA extracted, hydrolyzed and subequently analyzed by high pressure liquid chromatography as indicated in the Materials & Methods.

no d i f f e r e n c e and

in t o t a l

SV40-transformed

genomic

human

DNA methylation

fibroblasts

between

as m e a s u r e d

normal

by o u r

method. Our affect

results

the e x t e n t

fibroblasts. that

gives

the p u r i t y presence

sizes

viruses

It m u s t

above, affect

measure

by a r t i f a c t s

the

cells

cells

In a d d i t i o n ,

due

the e x t e n t

not

in h u m a n

have

of t o t a l

indicated genomic

t h a t the H P L C m e t h o d

by m o n i t o r i n g using

or if p u l s e

prove

for t h e

that

label may

in p r e c u r s o r

labels

post-DNA

are used

synthesis the

used

In a d d i t i o n ,

radioactive

to d i f f e r e n c e s

one m u s t

endonuclease

studies

of the D N A bases.

precise

does

DNA methylation other

Other methods

in 5 - m e t h y l c y t o s i n e .

restriction

genomic

of the D N A is c o n t r o l l e d

in the c o m p a r e d

period.

SV40-transformation

be e m p h a s i z e d

an a b s o l u t e

timing may miss

only

of t o t a l

of u r a c i l .

be h i n d e r e d

that

As m e n t i o n e d

transforming

methylation. here

indicate

label

pool the

methylation is f o u n d

The u s e of m e t h y l a t i o n - s e n s i t i v e analysis

1382

to m e a s u r e

DNA methylation

Vol. 102, No. 4, 1981

suffers sites

because

other

It also

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the m a j o r i t y

than those

should

defective

of DNA m e t h y l a t i o n

specific

be noted

that

although

in their m e t h i o n i n e

methionine-containing

their

endonucleases.

the P1 and P5 lines

metabolism

medium

sites m a y be at

for the r e s t r i c t i o n

(12,15-18),

are

in

DNA m e t h y l a t i o n

is s e e m i n g l y

unaffected. Although

our data do not

DNA methylation site-specific possibly

as a basis

changes

in m e t h y l a t i o n

have p r o f o u n d

possibility

implicate

effects

of changes

changes

in total

for S V 4 0 - t r a n s f o r m a t i o n

m a y occur w h i c h

on gene

cells,

would

transcription.

in s i t e - s p e c i f i c

genomic

of human

The

DNA m e t h y l a t i o n

remains

to be investigated.

ACKNOWLEDGEMENTS. This work was supported by grant CA27564 from the National Institutes of Health; The Council for Tobacco Research-USA, Inc.; The United Cancer Council, Inc.; The Cancer Research Coordinating Committee of the University of California; Grants from the Academic Senate, University of California, San Diego; and a Special Fellowship to R.M.H. from the Leukemia Society of America. E.S.D. is a NIH Post-doctoral Fellow and is supported by USPHS grant #AM07318-03. We stand indebted to Professor Jerry A. Schneider for his generosity.

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