Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy

Vol. 132, No. 3, 1985 November

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1143

BIOCHEMICAL

15, 1985

RIBONUCLEOTIDE REDUCTASE IN ASCITES TUMOUR CELLS DETECTED BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY G.

Lassmann,

8. Liermann, Koberling

A.

Central Academy

Received

of

September

1. and

P.

Lehmann, Langen

H.

Graetz,

of Molecular Biology, GDR, Robert-Rassle-StraOe

Institute Sciences of GDR-1115

10

Berlin-Buch

23, 1985

SUMMARY: Tyrosine radicals localized in the b12 subunits of ribonucleotlde reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-henoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of riboQ 1985 Academic nucleotide reductase in intact ascites tumour cells. Press,

Inc.

Ribonucleotide

INTRODUCTION: enzyme

of

cell

proliferation

having

and

a functionally 3+. oxobridged Fe ions

in the

in mammalian cells.

In

trum

from

conditions

tyrosine

essential

of 20-50

in comparison

cells,

has not

uetecteo

Abbreviation:

intact

been

RR and

to

modified

to the that

in

any

(l-6)

been

radicals wild-type.

in intact way,

The

the

by

as well

as

EPR doublet only

two

spec-

under

In these

cases the

enhanced

by

aim of

proliferating

tyrosine

enzyme

stabilized

recorded

was

a key

as a unique

in E.coli

however,

so far

is

1.17.4.1)

radical

of RR (5-11).

tyrosine

show

tyrosine

has

(EC

was recognized

cells,

of an overproduction of

(RR)

B2 subunit

radicals

concentration

paper

reductase

radicals

the

a factor

present

ascites of RR can

tumour be

by EPR.

RR - Ribonucleotide redutitase EPR - Electron paramagnetic

1137

resonance

0006-291X/85 $1.50 Copvrighi 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 132, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

h$ATERIALS AND METHODS: Cell preparation. Different samples of mrlich ascltes mammary carcinoma cells were taken from the intraperitoneal cavity of mice between 3 and 14 days after transplantation, thus varying in their proliferation rate from very fast to very slow (stationary state). After washing the cells in 150 mM NaCl an d centrifugation, cells were counted and a dense pellet (1.5.10 cells/O.3 ml) was pipetted into a cylindrical EPR quartz tube of 4 mm internal diameter and frozen to 77 K.-The ascites tumours SOV/lG% and 5A/lA were prepared in the same way. The time between removal and freezing of the cells was about 20 min. For treatment with 1 mM hydroxyurea the cells were incubated for 1 h in medium 199 and 4 % calf serum at 35OC before freezing. Tissue preparation. The solid forms of Ehrlich carcinoma, another mammary carcinoma (Ma-Ca 20177) and an ultraviolet-induced tumour (UVI 14306) were obtained by transplanting cells intramuscularly into the legs of mice. After 14 days the tumours (size about 1 cm) were excised, necrotic parts removed, the tumour tissue sliced and put into the EPR quartz tube. Enzyme preparation. The cells (taken 8 days after transplantation) were suspended in 15 mM imidazol buffer, pH 7.3; containing 15 mM NaCl, 1 mM dithioerythcitol, 1 rnlL KgCl2 and disrupted by 10 to 15 stroices %i t,il a Pot,tec-type homogenizer. The homogenate was centrifuged at 27,000 g for 30 min. The supernatant fluid was used as the crude enzyme extract. In some cases precipitation with acetic acid was done to prepare a dense protein pellet for EPR. Ribonucleotide reductase assay. IQe assay mixture contained in a final volume of 200 pl: 0.04 mM C-cytidine diphosphate (spec.act. 455 mCi/mklol ) ; 10 mM dithioerythritol; 1.5 mM ATP; 3 mM magnesium acetate; 6 mM sodium phosphate buffer, pH 7.0 and 100 pl of the The mixture was incubated at enzyme solution (l-2 mg protein). 37OC, usually for 60 min. Activity was linear up to 120 min; all assays were run in duplicate. The following steps (e.g. hydrolyzing of the nucleotides by snake venom) were done according to (12). Separation of cytidine and deoxycytidine was performed by paper chromatography in isopropanol/(O.l M boric acid)/conc. ammonium Radioactivity of the samples was determined with hydroxide (7:2:1). an LKB liquid scintillation spectrophotometer. The enzyme activity was calculated in nmoles deoxycytidine diphosphate formed per hour per mg protein. Protein content was determined according to (13). EPR spectroscopy. EPR spectra ‘vere recorded on a VARIAN E3 spectrometer in a cold-finger cryostat at 77 K with microwave power 63 mW or 200 mW and modulation amplitudes of 5, 10 or 12.5 gauss. The spin concentration was estimated at 20 mW by double-integration of spectra using a small computer KRS 4201 (Robotron, GDR) on-line A frozen ethanolic solution of coupled with the EPR spectrometer. a nitroxyl spin label with known concentration (determined optically) was used as a standard. RESULTS:

asci tes pronounced

Ribonucleotide t,umour

ccl 1s. Ehrlich

doublet

22 gauss

(Fig.

tyrosine

radicals

spectral

resolution

gauss is visible

reductase

in

ascites with

EPR spectrum

1 A,

C).

(2.3 of

(Fig.

In

cells

pid; 3 days the

1 A).

fast

carcinoma g = 2.004

with

the

aFter

two equivalent EPR

spectra

1138

proliferatin

g Ehrlich

dells

exhibit

and a splitting

highest

of

concentration

transplantation) ring-protons of this

a

type

of

a further

(5) were

of 5-6 found

BIOCHEMICAL

Vol. 132, No. 3, 1985

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gI

2.004

1. EPR spectra i-i%TT transplantation (63 erent

states

Ehrlich proliferation

-free

(200

of

Extract

of

A)

12.5

gauss;

gain

of mice

3 days

after

da s after

Cr 513

2.5.10

proteins from a cell (200 mlv; 5 gauss; gain

>.

Fig: 2. orgln at ascites sarcoma carcinoma enlarged

EPR spectra of mouse ascites tumour ells of different 77 K (63 mW; 10 gauss; gain 1.25.10 5 ); A) SOV/16 B myeloma, (4 days after transplantation, B) SA 1A ascites (4 days after transplantation), C) Ehrlich ascites (7 days after transplantation), D) as Fig.1 D with scan range for comparison.

and

were

to

site

of In

attributed the

h:2 subunit

ascites

cells

the

in other

g = 2.004 ponents

from at

g = i.956

2 A,D,C)

symmetric

RR an

and

g-factor.

tyrosine

and

tyrosine

radical

doublet

is the

remarkable

cells

additional End

isolated

enzyme

in the

(4),

active

RR.

without

tissues

in the

and

a stable of

at g = 2.00

spectrum

(5-9)

days 0)

),

3.2.10

cells

Fig.

ml;

acetic acid-precipitated Ehrlich ascites cells

in RR-overproducing

found

775K;

mW; 5 gauss;

transplantation

EPR spectrum

cells

carcinoma at

gain 5.10 >, 8)57 (200 ml4; 12.5 gauss; gain 2.5.10.),

transplantation after

ascites

of of

g-l.936

represents EPR spectra

Besides

EP'R spectruin appeers

a paramagnetic of 1139

this

type

the

centre have

been

at

of two cot>-

1 A,G,C with

often

doublet

consistin (Fig.

EPR

lines

underground

(14-16).

g = 2.025

dominating

and an

axial

attributed

Vol. 132, No. 3, 1985 to

non-hem

BIOCHEMICAL

iron-sulfur

complexes

superimposing

on

proliferating

state

cell-free

In

Lethods)

the

associated

the

2 0)

spectrum

is similar

tyrosine

from

with

radicals

of

of

ribonucleotide

ting

of

Ehrlich

tensity

of

the

activity

the

proliferation

radicals zymic and

of

the

tvi2 in

2 pH,

tumour after

Concentration

radicals

be

and

ascites

absent

a membrane-

of

their

EPR

cells

(4 days inhibitor

a specific

the

of the with

cells.

activity

doublet the

Tab.

of

tyrosine

prolifera-

1 shows

the

values

ascites

Although

cells.

requires

cells

on

tyrosine en-

active

subunits

All

with

and enzymic and

quiescent

3

7

13

2.8 + 0.3

1.4 2 0.3

0.6 + 0.3

1.1.

1.1 f ‘0.1

0.5 + 0.2

1140

and

and the

significantly

decrease

ascites

dependent

in-

only,

(PM)

Enzymic activit t; of RR (Relative units (nmoles deoxycytidine diphosphate /hr/mg protein)

1 D,C>,

as

the 1112subunit

transplantation) of

the

biochemically,

biochemically both

on

measured by EPR (Fig.

of Ehrlich

ratio,

activity

tumour

Ehrlich of

are

cells.

TABLE 1 Concentration of tyrosine radicals activity of RR in fast proliferating Age (days

to

to an amplitude

estimated

RR,

measured

a I:1

and

2.025

40 9: in comparison

radical

rate

reflect activity

about

reductase

ascites

tyrosine

the

and

the

sample (100 y:).

Dependence

state

on

the concentration

Ehrlich

about

g = 2.025

depend

is assumed

intact

leads

at

(see Materials

1 mM hydroxyurea,

scavenger,

control

untreated

origin

of

shoulder

at g = 1.936

proliferating

fast

radical

RR and

cells

sample is

to that

transplantation)

after

ascites

protein;

in this

of

1 E,C).

its

that

RESEARCH COMMUNICATIONS

does not

(Fig.

iron-sulfur

radicals

Incubation

of

The

line

doublet

components so

non-hem

(17).

of cells

spectral

tyrosine

of

left

extracts

1 D and

(Fig.

AND BIOPHYSICAL

de-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 132, No. 3, 1985

creasing

proliferation

ved

in (18)

also

parallel

to

may be

the

concentration

content

of

a higher

radicals.

degree

anaerobically radicals

(19)).

cscites

cells

and

ascites

cells

a high

also

cell

the

content

of tyrosine

(6,

residues

lo),

into

suffer

from

37 y: of the

cells

exist

of of

from

tumour

tissue,

however,

which

is also

RR in

ascites

other

Fir,.

mice give

tumours

The first from

in

comparison.

tyrosine

studies.

radicals it

lack

on

further

loo!< for

for

oxygen

tumours.

tyrosine

‘

14-day-old

that

influence

or

tyrosine

and

~1s to

tumours

different

elsewhere

considered

of

prompted

enzyme

value

of different

doublet

as obser-

may 5e due to a higher

be a subject

cells

in

the

enhanced

density

the

iyill

in solid

of

be

conditions

rcductase of

tur.iours

should

cells

observation

orciinary

The

of

of tyrosine

Therefore,

in ascites

Ribonucleotide direct

it

similar

(under

a decrease

cells

of activation

RR activity

of

Kl as observed

than

exhibit

tunours

oxygen

by rate.

Additionally,

ascites of

caused

decrease

in 3-day-old

subunits

K2

The

proliferation

radical

to

rate.

2 shows that

comparable

EPR

signals. In solid nppcars of

Ehrlich

(Fit;.

r,iicc,

3 C),

c,c:.

UV-intiuci?c’

a transplanted

this

typs

(20)

or nitrosyloyo~lo!~in

tissue,

hcvc

c.‘j.

been

in

45 and Walker

carcinosarcoma

NO-hemoprotein that

of the

in the

spectrum tyrosine

region

Pcssy melanona, shown)

was

ascitcs

not

of c~ = 2.00.

clearly

for

JAX

(15)

256 (15).

in Fig.

Also

and

2 B and

solid

the

22a,

due to the

of sarcoma

concentration

30 times both

EPX

tumour

neuroblastona

spectra

doublet

singlet

of the

larger

in the melanoma B12 and

1141

tunours

a transplanted

hepatoma

The spin

comparison,

detectable

C 1300

3 C is about

in Fig.

solid

nitrosylhemoglobin

in other

as

and

hepatoma

doublet

studiccl

as ~11

spleen

of

rat

and

in pentacoordinated (21)

13i c e ( 3.4) , in

in other

EPR spectrum

3 A z;T;:i !l) I P _...:., :: r‘c c :-r L 0 f

(?is.

reported

sarcoma

observed

manmary carcinoma

irc:::;2ccijv::ly

Lli:,!:iI.iT,

a different

from

than

overlap the Ilardingfrom

RR (not

melanin

Vol. 132, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EPR spectra of different tumour tissues of mice at 77 K 10 gauss); A) solid mammary carginoma Ma-Ca 20177 (20 days aker transplantation; gain 6.2.10 ), B) solid UV-light induce g tumour (UVT 14306) (14 days after transplantation; gain 6.2.10 ), C) solid E$rlich carcinoma (14 days after transplantation; gain 1.25.10 ). 3.

radicals.

Therefore,

to

in the

detect

DISCUSSION: well

as

is

the

dependence

from

sl;ate

concluded

Ascites

cells

radical

because

the

cells

may be

components psroilagnetlc

of

.

ions

are

spectra

suited

for

the

arising in the 1142

fron

of

Ehrlich of RR.

EPR studies

of the RR

clue to the the

L:cderground

absence

EPR spectruni

and 2.025, redox

on

hydroxyurea

radicals

of

as

activity

in intact

exception

at s.= 1.936

(5-9)

enzymic

influence

recognizable

With

visible

And

tyrosine

from

centres not

literature

EPR spectrum

is riell

centre

with

from the

and

observed

signal.

paramagnetic

.

EPR

originates

iron-sulfur

a non-heE

of

exceptionally

IJO-hemoprotcin

the

is difficult

present

tumours.

in comparison

latter

the

l?i: po ssibly

from

solid

of cells

tilat

carcinoma

from

of

EPG data

ascites

of

cast

From

proliferating it

a doublet

other

enzymes with of

i

greater

BIOCHEMICAL

Vol. 132, No. 3, 1985 sweep

range

of

this

system

cell

proliferation,

EPR spectra

ascites

from

is recommended

for

the

reductase

by

RESEARCH COMMUNICATIONS

cells

study

as activation

ribonucleotide radical

AND BIOPHYSKAL

of

(Fig. functional

by mitogens

cytostatic

2).

aspects

inhibition

or

with

agents

Therefore, of

of

properties

of

scavengers.

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