Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REDUCED LEVELS OF ADENOSINE DEAMINASE IN CHICK EMBRYO FIBROBLASTS TRANSFORMEDBY ROUS SARCOMA VIRUS Peter Laboratory

K. Chiang

and Giulio

L. Cantoni

of General and Comparative Biochemistry National Institute of Mental Health Bethesda, Maryland 20014 and David

A. Ray and John P. Bader

Laboratory of Tumor Virus Genetics National Cancer Institute National Institutes of Health Bethesda, Maryland 20014 Received

July

29,1977

SUMMARY: Adenosine deaminase activities in chick embryo fibroblasts were substantially reduced after infection and transformation by Rous sarcoma virus. Concomitant with the reduction in adenosine deaminase activities, the incorporation of exogenous adenosine into RNA species of the virus transformed cells was moderately increased. The significance between reduction in adenosine deaminase activity and malignant transformation by Rous sarcoma virus remains to be elucidated. INTRODUCTION Malignant oncogenic

transformation

viruses

is

and by a number of biochemical

accompanied

biochemical

changes

in chick

by Rous sarcoma virus water

cl),

decrease

tions

(2),

lower

hexose uptake protease

activity

activities

(8).

Copyright All rights

of a variety

of cells

by morphological changes.

and hyaluronic (7),

cyclase

activities

acid

synthesis

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

here

that

transformed

accumulation

cyclic

and decreased

We report

among these

embryo fibroblasts

in intracellular

adenylate

alterations

Notable

(RSV) are increased

by

of

AMP concentra(3,4), (5,6),

alkaline adenosine

increased increased

phosphatase deaminase

Vol. 78, No. 1, 1977

(EC 3.5.4.4)

BIOCHEMICAL

activity

substantially

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

these

RSV infected

cells

is

decreased. MATERIALS

AND METHODS

Chick embryo fibroblasts and similar cells transformed by different strains of RSV, were cultured in Eagle's minimal medium supplemented by 5% fetal bovine serum (5,6). Two days after replating, at which time growth rates and hence cell density were approximately the same, the fibroblasts were rinsed twice with Dulbecco's phosphate buffered saline, scraped off by a rubber policeman and suspended in 20 mM potassium phosphate, 250 mM sucrose, 0.1 mM dithiothreitol, The pooled cells were homogenized at 4oC by a pH 7.4, 4oC. ground glass homogenizer, and subsequently transferred to a glass centrifuge tube, frozen in liquid N2 and thawed. The supernatant, after centrifugation at 15,000 X g for 10 min, was used to assay adenosine deaminase activity by a method adapted from Gustin and Kemp (9). The assay medium in a final volume of 0.5 ml con ained 20 mM potassium phosphate, 1 mM EDTA, and 0.5 mM [8- 11 Cladenosine, pH 7.4, 370C. After 10 min of incubation, 50 1~.1 of 5 M formic acid was added to stop the reaction. The amount of [8-14C]inosine formed was determined by a SP-Sephadex column (9). Specific activity is expressed as wmoles of inosine formed per milligram of protein per hr; each value is the average of 3 to 4 determinations based on a linear relationship between enzyme activity and protein concentrations. The incorporation of adenosine and uridine into RNA species of infected and normal chick embryo fibroblasts were determined after 10 hrs of incubation with [2,8-3Hladenosine (10 pCi/ml; 30.4 Ci/mmol) and [U-14C]uridine (1 bCi/ml; 463.6 mCi/mmol), the RNA was extracted with 1% sodium dodocyl sulfate-phenol, precipitated by ethanol, sedimented in a 1530% sucrose gradient, and radioactivity determined. RNA sedimenting between 20s - 26s was adsorbed to oligo(dt) to select the polyadenosine containing species. RESULTS Table chick

1 shows the

embryo

activity

fibroblasts

by two RSV strains.

in

cells

sarcoma

infected

virus

decreased

to

(RSV-BH),

mental essentially

by the

It

conditions

or the that

is noteworthy the

unchanged

Bryan

levels (A.

deaminase

sells

after

of

adenosine

The activity

11 and 7% of

respectively.

of adenosine

and in similar

mation the

AND DISCUSSION in transfordeaminase

high-titer

strain

of Rous

Schmidt-Ruppin

strain

(RSV-SR)

of the that of acid

V. Bader,

337

non-infected under

similar

phosphatase J.

Kondratick

cells, experiremain and J. P.

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1. Adenosine deaminase activity in chick embryo fiborblasts infected with strains or mutants of Rous sarcoma virus (RSV). Specific activity is expressed as kmoles of inosine formed per milligram of protein per hr. Actinomycin D or cycloheximide were added at 2 Kg/ml of culture medium.

Cells

infected

with

Adenosine

deaminase

(specific

activity)

None RSV-BH RSV-SR

370 37O 37O

4.20 0.48 0.31

RSV-BH-Ta RSV-BH-Ta RSV-SR-T5 RSV-SR-T5

41° 37O 41° 37O

1.32 0.45 2.86 0.57

RSV-BH-Ta RSV-BH-Ta RSV-BH-Ta

41°-37O 41°-370 41°-37O

(6 hrs) (actinomycin (cycloheximide,

D, 6 hrs) 6 hrs)

1.44 1.62 1.32

RSV-BH-Ta RSV-BH-Ta RSV-BH-Ta

370+41° 370-41° 370-41°

(6 hrs) (actinomycin (cycloheximide,

D, 6 hrs) 6 hrs)

0.55 0.39 0.36

in

Bader, against

preparation).

large

Dialyzing

volumes

the

of buffer

did

not

activities

of adenosine

deaminase

the

extract

RSV-BH infected

enzyme

extract

from

expected normal able

normal

specific cells.

cells

Hence,

cells

Two mutants temperature-dependent

the

appreciably. cells

of adenosine

a virally inhibitor

adding

to the

enzyme

effect

production

of adenosine

specific

Also,

deaminase

induced

on the from

the

of dialyz-

deaminase

in

the

seems improbable. of RSV, RSV-BH-Ta transformation

at 41°C are morphologically

37OC cells

extracts

change

had no noticeable

activity

or non-dialyzable

infected

grown

from

enzyme

activity

are morphologically

and RSV-SR-T5, in

infected

non-transformed, transformed

338

induce cells. while

and exhibit

Cells at the

Vol. 78, No. 1, 1977

associated

BIOCHEMICAL

biochemical

are

cells

significantly

at 37OC,

to those

(Table

temperature

infected

are usually

in

uptake

hexose

within

when the

same cells

adenosine

deaminase

shifted

morphology

cells

reduced activities

were grown levels were

found

activity

This

increase

of adenosine RSV-BH-TA

reported

features

responsible

for

temperature Multiple

deaminase

adenosine

infected in

shifts forms

of

cells.

was observed from

the

presence

(5,6), changes

of mutant

infected

adenosine

deaminase

and alterations

isozyme

of

transformed

in cells

339

the

D, on

A small

increase 6 hours

37OC to 41°C. of actinomycin

in adenosine

when compared

the morphological

unaltered

by actinomycin

cells

of transformation

in

had no effect

changes

slow

shifting

essentially

deaminase

(10,11,12,13,14) patterns

of

Apparently,

are relatively

transformed after

these

observed

D or cycloheximide. activities

in

reversible;

By contrast,

by cycloheximide

was not

are

their

remained

activity

of trans-

are unequivocal

of RNA synthesis

synthesis

deaminase

shifting

cells

from

30 min, and increases

changes lose

activity

Inhibition

or of protein

after

These

switched

characteristic

synthesis

a few hours. the

are

within

to 41°C

infected

6 hours.

the

37'C

to 37OC,

adenosine

changes

(5,6).

from

RSV-BH-Ta

RSV-BH-Ta

and hyaluronic

within

41°C

after

with

observable

4 to 6 hours

cells

ing

these

transformed

37OC, morphological

formation

in

than

activity

1).

to

the

Nevertheless,

of wild-type

When cells

from

deaminase

at 41°C was markedly

cells.

higher

at which

similar

41°C

cultured

to non-infected

RESEARCH COMMUNICATIONS

Adenosine

changes.

in mutant-infected compared

AND BIOPHYSICAL

deaminase

to some other and may not

which

occur

be follow-

cells. have

been reported

adenosine

deaminase

were observed

(lo),

but

Vol. 78, No. 1, 1977

Table 2. of infected

BIOCHEMICAL

Incorporation and normal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of adenosine and uridine chick embryo fibroblasts.

Ratio RNA type

RSV-BH infected

7.0

8.3

Ribosomal

28s

6.7

7.7

Ribosomal

18s

7.6

9.1

4-7s

7.0

8.5

Oligo(dt)

selected

9.0

11.7

it

known whether

Low Mol.

Wt.

is not

the

isozymes

Concomitant

with

there

activity,

[3H]adenosine infected

(Table

incorporation

the reduction

all

could

of

The increase

deaminase.

rate

of adenosine

such as an alteration

in the rate intracellular

adenosine,

been eliminated.

that

at

uptake

the levels of adenosine

Adenosine with

the

disease

in the

there

that

of uptake

It

of

should

in the present

of a human combined by either

340

of

of adeno-

the

increased

factors, adenosine capacity be noted

was no competition

has received

is accompanied

level

phosphorylation

and uridine

deaminase

discovery that

used,

of

degradation

was due to other

or a change in the has not

deaminase

in adenosine

the possibility

incorporation

of

from the RSV-BH

to reduced

of the decrease

However,

sine

all

some of them.

in adenosine

the RNA species

2).

involve

in the incorporation

be attributed

as a result

here

deaminase or only

was an increase into

cells

adenosine

changes observed

of adenosine

RNA

[3H]adenosine/[14C]uridine

Normal

Total

into

of here

between

the

investigation.

much attention

recently

immunodeficiency an absence of or much

BIOCHEMICAL

Vol. 78, No. 1, 1977

reduced

levels

found

that

from

patients

of

the

levels with

consistenly

lower

(18,19).

adenosine

than

xanthine

for

use in

reduced

levels

of

in

the

to

adenosine

in

purine

adenosine cells

deaminase

It

in

plays

from a key

and the salvage

deaminase

transformed

was

subjects

in

the

production

pathway affect

leukemia

normal role

was also

lymphocytes

lymphocytic

lymphocytes

nucleotides the

(15,16,17).

chronic

deaminase

of adenine

parameters

deaminase

untreated

Adenosine

metabolism

remains

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

normal of hypo-

(20,21). other

by Rous sarcoma

How

metabolic virus

be elucidated.

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Bader, J. P., Ray, D. A., and Brown, N. R. (1974) Cell, 3, 307-313. Otten, J., Bader, J. P., Johnson, G. S., and Pastan, I. (1972) J. Biol. Chem. 247, 1632-1633. Anderson, W. B., Johnson, G. S., and Pas-tan, I. (1973) Proc. Natl. Acad. Sci. U.S.A., 70, 1055-1059. Gidwitz, S., Weber, M. J., and Storm, D. R. (1976) J. Biol. Chem., 251, 7950-7951. Bader, J. P. (1972 J. Virol., 10, 267-276. Bader, J. P., Lew, M. A., and Brown, N. R. (1976) Arch. Biochem. Biophys., 175, 196-208. Unkeless, J. C., Tobia, A., Ossowski, L., Quigley, J. P., Rifkin, D. B., and Reich, E. (1973) J. Exp. Med., 137, 85-111. Bader, A. V., Bigelow, M., and Kondratick, J. (1975) Abstracts Ann. Meeting Amer. Sot. Microbial., 249. Gustin, N. C., and Kemp, R. G. (1976) Anal. Biochem., 71, 527-531. Jenkins, T., Rabson, A. R., Nurse, G. T., Lane, A. B., and Hopkinson, D. A. (1976) J. Pediat., 89, 732-736. Hirschorn, R., and Levytske, V. (1974) Cell Immunol., 12, 387-395. J. R. (1968) Biochim. Biophys Acta, Ma, P. F., and Fisher, 159, 153-159. Edwards, Y. H., Hopkins, D. A., and Harris, H. (1971) Ann. Hum. Genet., 35, 207-219. Van Der Weyden, M. B., and Kelley, W. N. (1976) J. Biol. Chem., 251, 5448-5456. Giblett, F. R., Anderson, J. E., Cohen, F., Pollara, B., and Meuwissen, H. J. (1972) Lancet, 2, 1067-1069. Van Der Weyden, M. B., Buckely, R. H., and Kelley, W. N. (1974) Biochem. Biophys. Res. Commun., 57, 590-595. Van Der Weyden, M. B., and Kelley, W. N. (1977) Life Sci., 20, 1645-1650.

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Vol. 78, No. 1, 1977

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Tung, R., Silber, R., Quagliata, F., Conklyn, M., Gottesman, J., and Hirschorn, R. (1976) J. Clin. Invest., 57, 756-761. Meier, J., Coleman, M. S., and Hutton, J. J. (1976) Br. J. Cancer, 33, 312-319. Green, H., and Chan, T.-S. (1973) Science, 182, 836-837. Mills, G. C., Schmalstieg, F. C., Trimmer, K. B., Goldman, A. S., and Goldblum, R. M. (1976) Proc. Natl. Acad. Sci. 73, 2867-2871. U.S.A.,

342