Increased incorporation of aspartate and decreased incorporation of orotate in fibroblasts from Lesch-Nyhan patients as revealed by autoradiography

Vol.

96, No.

October

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

16, 1980

INCREASED

INCORPORATION

FIBROBLASTS

OF ASPARTATE

FROM

by

LESCH-NYHAN

Amelia

de

de

la

August

de

AS

and

de

4,

OF OROTATE

IN

BY AUTORADIOGRAPHY

Santiago

Grisolia

Citol6gicas

Ahorros

Saboya

INCORPORATION

REVEALED

Investigaciones

Caja

Amadeo

DECREASED

PATIENTS

Martinez-Ram&

Instituto

Received

AND

1071-1076

de

Valencia

Valencia

(Spain)

12,198O

SUMMARY While it has been known for many years that tissues from Lesch-Nyhan --patients are deficient in hypoxanthine-guanine phosphoribosyl transferase, it has been demonstrated more recently that erythrocyte lysates show an increase in the levels of enzymes which participate in "de novo" synthesis of pyrimidines. Also, we were able to demonstrate, using autoradiography, an increased rate of incorporation of aspartate into the nucleus of intact cultured fibroblasts from a Lesch-Nyhan patient. We have now confirmed this finding. In this paper we show increased incorporation of aspartate into the nucleus of cells from three other patients. Interestingly, the incorporation of orotate is decreased in the fibroblasts from the four patients studied. The incorporation of leucine is the same in Lesch-Nyhan fibroblasts as in normal fibroblasts. The significance of these findings is briefly discussed.

The cy

(2)

best in

These

molecular

hypoxanthine

patients

On

these

patients

the

other

hand,

patients

carbamyl tient in

the

methods,

HGPRT.

The Although

and

the

(1)

transferase

in

the

increased

of

"de

of

novo"

is

the

(HGPRT)

activity level

increased

has

been of

and

more

an

increased

aspartate

much

syndrome

ribose

(E.C.2.4.2.8) phosphate

phosphoribosyl

production

deficien-

pyro-

pyrophosof

purines

charac-

(3,4). it

fibroblasts

Lesch-Nyhan

increase

metabolism

(5,6),

aspartic (7).

to

of

in

radiographic

an

the

contribute

alterations Nyhan

have

the

phosphoribosyl

(E.C.2.7.6.1);

should

teristic

in

guanine

also

phosphokinase pate

defect

found

recently

that

pyrimidines

in

erythrocyte

recently

attention

have

been

incorporation in

from

we

intact this

the

been

given,

to

almost and

a number from

show,

auto-

precursors

a Lesch-Nyhan entirely

properly

of Lesch-

using

pyrimidine from

were

are lysates

able

fibroblasts patient

has

of

there

pa-

deficient so,

to

the

de-

0006-291X/80/191011-06$01.00/0

1011

Copyright 0 1980 b.vAcademic Press, Inc. All rights of reproduction in any form reserved.

Vol.

96, No.

3. 1980

ficiency the

of

the

increase

is

most

which

BIOCHEMICAL

enzyme

in

the

interesting uses

to

three

other

HGPRT

deficiency

these

order

to

kindness

the

detect

2.4.2.10.)

Also,

which

is

with

the

Prof.

Nyhan

typical

high we

have

in

one

of

at

incorporation

of the

the

patients, cell

major

below,

fibroblasts

last

steps

well

with

the

in

fibroblasts

of

of

one

from

as

aspartate

phosphoribosyl

but

pathway. the

incorporation

level

pathway

manifestations, We show

of

these intact

tested

syndrome.

orotate

COMMUNICATIONS

biosynthetic

have

clinical

in

the

another

purine we

tested

variations

involved

demonstrated

common

the

RESEARCH

deficiency

involves

the

possible

mental

only

Lesch-Nyhan

methods, patients.

the

BIOPHYSICAL

not

of

showing of

autoradiographic

it in

subjects

to pathway

because

the

and

pyrimidine

intermediates

Thanks

from

HGPRT

AND

erotic

as aid

acid

transferase pyrimidine

the

of

in (E.C.

biosynthe-

sis.

MATERIALS AND MET ODS L-(2,3-3H) aspartic acid (12 Ci/mmol), L-(4-3H) leucine -(flfi=lTaa-(+ 5 H erotic acid (15 Ci/mmol) were from the Radiochemical Center, Amersham. The bovine fetal serum and Eagle culture medium, minimum essential medium (MEM) from Flow Laboratories (Irvine, Scotland). Cycloheximide was from Sigma, and other chemicals used were of the highest purity available. Cell cultures. The fibroblasts designated line V.G. were cultured by the explant method from a skin biopsy of a five-year old child with the Lesch-Nyhan syndrome. The lines D.G., J.R.R. and C.W.W. were fibroblasts from other patients with the same disease, 8,14 and 16 of age respectively, and were from the laboratory of Dr. Nyhan, (Department of Pedriatics, La Jolla) University of California in San Diego. The cells were grown in monolayer in 25 ml Falcon flasks at 37°C in Eagle's MEM supplemented with 20 % bovine fetal serum, penicillin (100 I.U./ml) and streptomycin (100 ug/ml). The control human fibroblasts were obtained from the skin of a normal child and were cultured under the same conditions. Labeling of the cells. To cells grown on cover-slip3 for 48 h after the last passage (10,15), was added fresh medium containing ( H) aspartic acid, 5 u Ci/ ml (4 PM) for 1 h. Cycloheximide (200 pg/ml culture-medium) was added 10 min before the isotope3 was also present3in the medium throughout the incubation period with ( H aspartate. When ( H) leucine was used, 2 LICi/ml (4 uM) were added to the cu ture medium for 1 h. In another expesiment, cyclohexiS mide was added with the same conditions as described for ( H) aspartate. ( H) erotic acid (10 pCi/ml) (8 uM) was added and the pulse was the same as for the other isotopes. The autoradiograms were prepared using the "stripping film" (8) technique with Kodak AR10 film, exposed for 8 days at 4°C. After developing with D-19 (Kodak) and fixing with Agefix (Agfa), the cells were stained with a citratebuffered Giemsa's solution.

RESULTS as

AND

evidenced

DISCUSSION by

the

The lack

of

cells

used

in

incorporation

this of

1012

study (3H)

show

hypoxanthine

a deficiency and

of

of

HGPRT

(3H)

gua-

Vol.

96, No.

BIOCHEMICAL

3, 1980

Fig. 1. Incorooration a-c acid in the were as described in blasts.

nine.

We have

higher

incorporation

mal

fibroblasts,

cation. and

under

these of

this,

throughout

us Nyhan and

to

results

(7).

are Fig.

fibroblasts as

previously

the

expected,

[3H)

large the

in

was

three

RESEARCH

of

protein

1 illustrates

COMMUNICATIONS

to

tested

in

control

is

a larger

we

in

the

nuclei

with

the

incorporation

cytoplasm.

1013

of

have

of . marks

demonstrate

Also, in

permit

with

in

nor-

quantifi-

cycloheximide,

the

incorporation.

used

cycloheximide

and first

in case

(3H)

aspartate

as

i7lustrated

the

a

than

not

inhibited

quantitate

obtained

number

did

now,

fibroblasts

to

fibroblasts marks

both

a larger

possible

was

possible

those

was

Lesch-Nyhan

radioactive

cases

to

it

of

incorporation similar

there

aspartate

synthesis

quantitate

than

while

it

the

very

that

number

conditions in

BIOPHYSICAL

by normal and Lesch-Nyhan syndrome fibroblasts of f3H) presence of cycloheximide. The experimental conditions the text. a) normal fibroblasts b) Lesch-Nyhan fibro-

of

Therefore,

Because

The

shown

AND

nucleus

the

cytoplasm. studied in

by Lesch-

in than

Fig.3 in

the

Vol.

96, No.

3. 1980

Fig.2. Incorporation orotlc acid. The a) normal fibroblasts

BIOCHEMICAL

by normal experimental b,c,d,e)

NUCLEUS

:2&f 3

in the scribed fibroblasts.

AND

BIOPHYSKAL

RESEARCH

COMMUNICATIONS

and Lesch-Nyhan syndrome fibroblasts conditions were as described in the Lesch-Nyhan syndrome fibroblasts.

CYTOPLASM

f3H)

TOTAL

Number of qrains over the nucleus, cytoplasm and Lesch-Nyhan fibroblasts after incorporation presence of cycloheximide. The experimental in the text. N) normal fibroblasts 1,2,3,4)

1014

of text.

and entire cell in 100 of (>H) aspartic acid conditions were as deLesch-Nyhan syndrome

Vol.

0 4

96, No.

O

BIOCHEMICAL

3, 1980

CYTI 3PLASN

NUCLEUS

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I-

TOTAL

2

Fig. 4. Number of grains over the nut' leus, cytoplasm and enfire ccl' normal and ter incorporation of ("H) leuc, experimental conditions were as descr, ibed in the text. N) normal f, 1,2,3,4) Lesch-Nyhan fibroblasts. Number of grains over entire,cell asts after incorporation of ("HI were as described in the text. fibroblasts.

Nyhan

Following

the

(3H)

leucine

to

tions,

was

indeed

incorporation of

confirm

into

cycloheximide

using

very

low as

incorporation with As

there

Again,

in are

was this

proteins.

The

incorporation

almost

complete

phosphoribosyl

is

in

shown

leucine in

case,

there

is

Figs.

2 and

their

bulk

leucocytes

lack

have

there

5,

been are

no and

when

contain

1015

incorporation under

leucine

to

those

in

the

in

not

the

the

controls. the

an

erythrocytes slightly

to

presence by

incorpo-

extent

of

Also,

as

cytoplasm.

incubated

the

due

the

with

Lesch-Nyhan in

condi-

Also,

in

in

were

or

was

quantitate

of

of

our

and

differences

since

normal

enzyme.

able

was

conLesch-

incorporation.

with

particularly shown

of

cells

incorporation

been

(3H)

labeling

incorporation

interpret, has

aspartate,

the

biosynthesis

a preferential

of

of

of

cells

less

the to

4,

Lesch-Nyhan

surprisingly

checked

pyrimidine

we

Fig.

the

transferase while

in

we

incorporation

mostly

in 100 e. The roblasts

in 100 normal and Lesch-Nyhan erotic acid. The experimental N) normal fibroblasts 1,2,3,4)

Horecker,

reflecting

activities

difficult

Dr.

the

specific

illustrated

acid,

patients,

that

between

expected,

of

resulted

and

ration,

sults

suggestion

34

(3H)orotic

fibrob7asts.

nucleus. increase

These in

of lower

re-

orotate

Lesch-Nyhan levels

of

the

Vol.

96. No.

BIOCHEMICAL

3, 1980

We do not enzymes

for

Lesch-Nyhan

know the mechanisms biosynthesis

patients.

The new finding

in

this

phosphoribosyl

paper,

of

these

always

phate,

and in a thorough study

Erlich

ascites

ferase

is

tumors

activated

Obviously,

of precursors

only tients, those

in intact

but may also

cells

leading

it

in orotate believed

in phosphoribosyl

pyrophos

phosphoribosyl

trans-

as of incorpora-

of biosynthesis

patients.

Such a study

metabolic

alterations

functional

and of may not

of these

alterations,

manifestations.

1.

Lesch,

2.

Seegmiller, J.E., 155, 1682-1684.

3.

Fox,

W. (1964)

4.

Torrelio, B.M. 87, 380-387.

5.

Beardmore, Med., 81,

6.

Kennedy,

J.

7.

MartinezBiophys.

RamBn, A., Rubio, V. and Grisolia, Res. Commun., 90, 333-337.

8.

Schmid,

9.

Shoaf, W.T. 45, 796-802.

Rosenbloom,

and Kelley, and Paz,

T.D., 43-52. (1978)

W. (1963)

Meade,

W.N.

Am. J. F.M.

(1971)

J.C.

Qtogenetics, M.E.

Med.,E,

gift of three cell lines the manuscript, and I. technical assistance.

Biochem.

and Kelley, Biophys.

2, (1971)

561-570.

and Kelley, Am. Int. --_

M. (1979)

Biochem.

and Jones,

pa-

particularly

REFERENCES

I.H.,

in

is necessary,

the pathways

of these complex

as well

ACKNOWLEDGMENTS We wish to thank W.L. Nyhan for the from Lesch-Nyhan patients, F. Thompson for help with Salazar, F. Rip011 and M.J. Argu116 for their expert

M. and Nyhan,

that

biosynthesis

of enzyme levels

(E.C.4.1.1.23.)

in clarifying

to behavioral

generally

orotate

in

of orotate,

a decrease

is

of

and others

of pyrimidine

study

of both

obviously

in activity

pyrophosphate.

in cells

help

However,

has been shown that

decarboxylase

on the

glance

enzymes

increase

incorporation

at first

and thorough

of pyrimidines

shed light

of the

COMMUNICATIONS

by ourselves

show an increment

by phosphoribosyl

of orotate

utilization

it

an extensive

particularly tion

(9)

the

of decreased

activity.

patients

RESEARCH

for

demonstrated

indicates

transferase

BIOPHYSICAL

responsible

pyrimidine

demonstrated

the cells

AND

W.N.(1967) Med.,

-,74

Biophys. W.N.

424-433. Res.

(1973)

Res Commun.,

Science,

Commun.,

J. Lab.

Clin.

8&, 653-658.

S. (1979)

Biochem.

175-193. Biochem.

1016

Biophys.

Res.

Commun.,