Hydroxyurea inhibits degradation of internalized epidermal growth factor in HeLa cells

Vol. 109, No. 1, 1982 November 16, 1982

BIOCHEMICAL

AND

BlOPHYStCAL

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202-209

HYDROXYUREA INHIBITS DEGRADATION OF INTERNALIZED EPIDERMAL GROWTH FACTOR IN HELA CELLS Hidetaka

Masuda*,

Mikio

Yamamoto,

Kiyoshi

Inokuchi*

and Hideya

Endo

Medical Institute of Bioregulation, *Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received

October

8, 1982

growth factor stimulates DNA synthesis in cultured SUMMARY: Because epidermal five inhibitors of DNA synthesis were tested in HeLa cells to see cells, whether the inhibition of DNA synthesis has any effect on the metabolism of the growth factor. Among these, only hydroxyurea inhibited the degradation of 1251-labeled epidermal growth factor strongly. The reversal of hydroxyureainduced inhibition of DNA synthesis by deoxyribonucleosides did not result in a recovery from the inhibition of the degradation. From these findings, it might be concluded that the inhibitory effect of hydroxyurea on the degradation is distinct from that on DNA synthesis.

been

EGF stimulates

DNA synthesis

suggested

the

peptide

that

fragment

whether

metabolism,

which of cultured

inhibition

cells (6-8),

EGF but

internalization

biological

the

steps

inhibitors activity

types

(1,2).

EGF on DNA synthesis

might

be mediated

proteolytic

of

as a second

of cell

cleavage

messenger

(3).

of DNA synthesis

was extensively

of DNA synthesis

other

from

complexes

us to test

internalized

of

originating

hormone-receptor

variety

effect

in a variety

investigated

(l-5).

We report

markedly revealed

decreased no detectable

and that

no similar

of DNA synthesis. of HU other

These than

the

actually 125

using here the

This

that rate

hypothesis affect

HU, known

effect,

however,

findings

also

by a

prompted EGF

I-labeled

on both

has

internalized

EGF in a wide as

of degradation

effect

effect

the

It

binding

an inhibitor of the and

was observed suggested

by the

a new

on DNA synthesis.

MATERIALS AND METHODS The following DME, calf serum and trypsin were obtained from Gibco. materials were from Sigma.: HU, arabinosyl cytocine, cycloheximide, actinomycin D, mitomycin C, 5-fluorodeoxyuridine, dAdo, dGuo, dCyd, bovine EGF=epidermal growth factor; DME=Dulbecco's Abbreviations: medium; HU=hydroxyurea; BES=N,N-bis-(2hydroxyethyl)-2-aminoethane 0006-291X/82/210202-08$1.00/0 Copyright All righrs

0 1982 by Academic Press, Inc. in any form resewed.

of reproduction

202

modified

Eagle's sulphonic

acid.

Vol. 109, No. 1, 1982

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

serum albumin and BES. [3H]dThd (20 Ci/mmol), [3H]Urd (40 Ci/mmol) and [3H]Leu (126 Ci/mmol) were from New England Nuclear. Bleomycin A2 was a gift from Nippon Kayaku Co., Japan. Mouse EGF was from Collaborative Research. Na1251 (15.6 mCi/pg I) was from the Radiochemical Center. Tissue culture multiwell plates were purchased from Falcon Labware. EGF Binding Assay: HeLa cells were grown in DME plus 10% calf serum in a 5% CO2-95% air at 37O. For experiment, 1.8 x 105 cells per well were inoculated into 17 mm multiwell plates. After 48 hr incubation, confluent monolayers of cells were washed twice with binding buffer consisting of DME containing 1 mg/ml bovine serum albumin and 50 mM BES adjusted to pH 6.8. After a 30 min of preincubation with or without HU, the cells were incubated for the required time intervals at 37" with 0.5 ml binding buffer containing 3 ng of 1251-EGF in the presence or absence of HU. 1251-EGF (specific activity 51863 cpm/ng) was prepared according to the chloramine-T method (9). After incubation, the cells were washed 4 times with cold binding buffer, solubilized with 0.5 ml of 0.5 N NaOH for 1 hr at 37" and cell-associated radioactivity was determined. Nonspecific binding was less than 3% of the total. All data were corrected for nonspecific binding. Measurement of Down Regulation: HeLa cells in confluence were incubated at 37' with 6 rig/ml of unlabeled EGF with or without HU. At the indicated times, the cells were washed twice with binding buffer, and 0.5 ml of binding buffer containing 3 ng of 1251-EGF was added per well. The cells were incubated for 1 hr at 37', and cellassociated radioactivity was measured. Dissociation of Cell-associated 1251-EGF: After incubation with 1251-EGF at 6 rig/well for 1 hr at 37', the cells were washed with binding buffer and 1 ml of fresh binding buffer was added per well with or without HU. The cells were incubated at 37". At the indicated the remaining cell-associated radioactivity was determined. times, Incorporation of [3H]dThd, [3H]Urd and [3H]Leu: The cell were incubated for 4 hr atmith 0.5 ml binding buffer containing [3H]dThd (lflCi/ml), [3H]Urd (1.5 J.&i/ml) or [3H]Leu (3uCi/ml) and the cells were washed and removed with various agents. After the incubation, The average viable cell number per well was 5.3 x 105 cells. 0.25% trypsin. After the centrifugation at 3000 rpm for 10 min, acid-insoluble radioactivity was measured. Effect of Various Agents on Degradation of 1251-EGF: HeLa cells in confluent monolaver were incubated with 1251-EGF at 3 rig/we 11 for 1 hr at 37". The cells were subsequently washed 4 times with binding buffer. The cell-associated radioactivity present at this time was taken as One ml of binding buffer containing the the initial zero time control value. The cells were incubated for 4 hr at 37", various test agents was then added. washed 4 times with binding buffer and cell-associated radioactivity was measured.

RESULTS The time absence

course

of HU are

at 1 hr was not with effect for below

HU led of

of

given

affected

125 I-EGF in Fig.la.

1 or 6 hr.

The initial

20 mM of

HU although

to HeLa cells

The initial

by 20 mM HU.

to an increase HU concentration

binding

on

125

binding it

I-EGF

binding

However,

in cell-associated binding

was not

was suppressed

in

the

125 I-EGF

of

continued

incubation

radioactivity. to HeLa cells affected

presence

by the

or measured up to 6 hr

Fig.lb after

shows the incubation

concentrations

by 80 to 60% at higher

concentra-

Vol. 109, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

i

200)

Time,

f-e%

’

0 0.5

1

hr

3

’

’

2

5

10

Hydroxyurea.

’

50

20

ml-4

w Effect of HU on lz51-EGF binding to HeLa cells. After 30 min preincubation at 37" with or without HU, cells were incubated for the indicated periods at 37" with 0.5 ml binding buffer containing 3 ng of 1251-FGF in the presence or absence of HU. Cells were washed and solubilized for the measurement of radioactivity. Each point represents the mean of three determinations. a) b)

Time course of 1251-EGF presence (0) of HU (20 Effect of HU concentration associated radioactivity incubation at 37' with concentrations of HU.

binding to HeLa cells mM). on 1251-E(;~ binding was determined after 1251-EGF in the presence

in

the t0

1 hr of

absence

HeLa cells. (0) or 6 hr various

(0)

or Cell(0) of

y 400 2 “0 =B d 5 0 n 8 BA

300

200

100

0

1

3

4

Time,‘hr

u Down regulation of EGF receptors by KF in the absence or presence of HU. The cells were incubated at 37" with 6 rig/ml of unlabeled EGF in the presence or absence of HU. At the indicated times, cells were washed and 0.5 ml of binding buffer containing 3 ng of 1251-EGF was added per well. After 1 hr of incubation, ceil-associated radioactivity was measured. Each point represents the mean of duplicate determinations. HU concentrations are: O,none; l ,20 mM;A,50 mM; n ,lOO mM;O,150 IIN.

204

loolxl

BIOCHEMICAL

Vol. 109, No. 1, 1982 tions

of 50 to

increased

with

associated 125

100 I&. doses

125 I-EGF

I-EGF

to cell

EGF receptors 6 rig/ml

the

induced

surface

obtained cell-associated

regardless

of the

presence

inhibit

Fig.3 in

the

shows

absence

rapidly

with

half

life

of

incubation

rate

internalization

the

time

as observed

life

period,

the

without

of HU, the

=

increase

with

in the

binding

decreased These

of

was 30 to

and by subsequent in a similar

findings

suggest

manner that

about

In

the

50 min.

absence,

the

In the

presence,

When HU was removed radioactivity

from

was rapidly the

125

of cell-associated

reversible

the

radioactivity however,

culture

decreased nature

decreased

wells at a of

this

apparent at

HU effect.

600

E L

Time,

Each

hr

Dissociation of cell-associated ‘*‘I-EGF. After incubation with 1*5Icells were washed and 1 ml of fresh binding 6 rig/well for 1 hr at 37’, was added per well with (0) or without (0) 50 mM of HU. At the indicated binding buffer was removed and remaining radioactivity was determined. After incubation for 4 hr at 37” with HU (50 mM), binding buffer containing HU was replaced by fresh binding buffer without HU. After further incubation for the indicated time intervals, the cell-associated radioactivity was measured. point represents the mean of duplicate determinations.

205

4 hr

similar

oi .E .E 400

(A):

HU

I-EGF

E

m EGF at buffer times,

of

EGF at

125 I-EGF

of

binding

(10)

unlabeled

control

of HU.

in cell-

down regulation

of dissociation

HU, indicating

m z” “0

the

of radioactivity

step.

of HU. of

7 hr.

shows

no preincubation

course

and presence

was about

Fig.2

or absence

amount

due to an increase

radioactivity

the

a half

Thus,

1 hr of preincubation

from

the

not

5 up to 100 mM.

or absence

incubation,

dose

the

by HU was not

After

RESEARCH COMMUNICATIONS

period,

receptors.

presence

value

6 hr binding

of HU from

by EGF.

in the

40% of

After

AND BIOPHYSICAL

Vol. 109, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Hydroxyurea,

RESEARCH COMMUNICATIONS

mM

m Effect of HU concentration on dissociation of cell-associated I251-EGF and [3H]dThd incorporation. Cells were preincubated with 1251-EGF and washed as described in Fig.3. After incubation for 4 hr at 37' in 1 ml of binding buffer containing the various concentrations of HU, cell associated-radioactivity was measured (0). In a separate group of assays, cells were incubated for 4 hr at 37' with 0.5 ml binding buffer containing [3H]dThd (luCi/ml) and the indicated concentrations of HU (A). All the values were the mean of triplicate determinations.

Dose response incorporation greater

curve

into

than

the

2 mM of

ated

radioactivity

that

HU concentrations

125 I behavior

(Table

I).

the

the degradation

on the

its

coupled

under the

into

which

reversal

the

effect

125

at higher

I-EGF

for

the

DNA synthesis.

the

effect

inhibition

HeLa cell

equivalent

t3H]dThd

of HU-induced

inhibition

of DNA

inhibition

the

of about On the

(20, in

HU

to 59.4% by the

was observed.

even

of

DNA was 7.9% of

at a concentration

conditions

206

be noted

of HU to inhibit

whether

concentrations

maintained

cell-associ-

80% affected

action

with

with

should

by about

Although

HU alone

of

It

and was recovered

(7,ll). with

remaining

inhibitory

incorporation

was fully

the

to determine

20 mM HU alone

125 I-EGF

of

that

was actually

of HU observed of

Thus,

with

degradation

The treatment

up to 100 mM.

implies

was obtained

deoxyribonucleosides recovered.

drug

investigated

of

1251-EZGF and t3H]dThd

DNA synthesis

This

[3H]dThd

incorporation

effect

of the

of

in Fig.4.

in an increase

deoxyribonucleosides

0.5 mM, no effect hand,

depicted

be unrelated

presence

degradation

inhibited

of degradation

of three

of dThd

doses

we further

in the

addition

HU resulted

slightly.

might

on inhibition

control

were

which

only

Therefore,

HU on the

cells

with

EGF degradation

synthesis

of

other

50 or 100 mbi) on presence

incorporation

of three was greatly

of DNA synthesis

did

Vol. 109, No. 1, 1982

Table

I.

BIOCHEMICAL

Effect of Various and Degradation

agents

AND

Agents on DNA, 1251-EGF

of

[3H]dThd incorp.*

100

0.5 ml4 1.0 mM 1.5 mM 20.0 mM 50.0 mM 100.0 mM 20 mM+3NdR+ 50 mM+3NdR+ 100 mM+3NdR+ 3NdR+ 5-fluorodeoxy uridine 0.01-50 mM arabinosyl cytocine 10-2-10-6 M mitomycin C 10-6-5x10-4 M bleomycin A2 10-6-10-3 M actinomycin D 10 ug/ml cycloheximide 200 fig/ml * ** + All

not

control

% of initial value 100

98.7 89.4 64.4

14.9 15.0 15.9 16.6 37.1 51.3 57.8 37.1 52.4 57.6 16.3

12.1 10.8 9.2

90.5-3.1

16.0-13.7

22.1-3.0

14.7-15.4

98.2-15.0

15.1-13.1

91.6-68.5

14.5-12.6 14.6 8.1

of effect

of DNA synthesis, C and bleomycin

mitomycin

degradation

process highly

of EGF might

14.6

the

of

arabinosyl

125 I-EGF that

be independent incorporation

on EGF degradation

The direct was less

of

not at

the

from

of 125 I-EGF.

of ULI on degradation

A2, did

suggested

concentrations.

revealed

1251-EGF

remaining**

6.4

alteration

inhibitors

inhibit

Synthesis

[3H]Leu incorp.*

100

58.0 23.2 16.0 7.9 7.6 5.8 59.4 29.4 27.5 86.4

Protein

Control values of incorporation of [3H]dThd, [3H]Urd and [3H]Leu were 52697 f 1609, 56843 f 1900 and 66019 f 804 cpm/lO6cells res ectively. The initial zero time radioactivity was 45121 f 2530 cpm/lO t cells. 3NdR represent the mixture of dAdo (0.1 mM), dGuo (0.1 mM) and dCyd (1wM). the values were the mean of three determinations.

produce

findings

RESEARCH COMMUNICATIONS

RNA and

[3H]Urd incorp.* % of

none hydroxyurea

BIOPHYSICAL

cytocine, exhibit

that

[3H]Urd correlation probable,

5-fluorodeoxyuridine, any significant

effect

concentrations

tested.

the various inhibitory

effect

of HU on the

on DNA synthesis. and [3H]Leu between since

no effect. 207

Other

both

into these

on These

degradation

HU was also

shown to

HeLa cells

at high

activities

and the

actinomycin

effect

D and cycloheximide

Vol. 109, No. 1, 1982

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION From this internalized

study EGF.

The degradation protease ketone the

effect

inhibitors,

leupeptin ester

It

energy

required

metabolic

is

also

energy

cyanide

the

had little

containing

both

podophyllotoxin,

are

unlikely

of

HU-induced

such

(9)

the

to be,

HU should

inhibited

the

a useful major

can be reversed

and in vitro

inhibitors

azide

of

or sodium

standard

medium

colcemid,

EGF degradation

(13).

and It

of microtubules with

might

giving

rise

lysosomes.

be caused

that

seems,

occurs

by a direct with

(15,16)

some kinds

or some tertiary

(17).

the clear.

of

of

vinblastine,

chloroquine

amines

part

however,

of metabolic

of EGF in the

manner

(14),

agent

by inhibiting

in vivo

sodium

vesicles

in a similar

ambiguity

chloromethyl

(9).

endocytic

investigating

become

EGF, since

DNA synthesis

of

lysomotropic

to make these

a number

depolymerization

as ammonium chloride

and other

both

of

of

(unpublished),

EGF, since

of EGF degradation

function

We are now further activity

fusion

kinds

production

to inhibit

HlJ causes

by several

the

degradation

acid

undiscovered.

experiment

as colchicine,

known

inhibition

on lysosomal

of agents

such

also

that

to an inhibition

on the

so far

probably

B was observed

of

and amino

agents,

(4,9)

HU inhibits

process

or tosyl-L-lysine

such as dinitrophenol,

influence

Antitubulin

however,

that

degradation

glucose

was

(K!), acid

cathepsin

production,

degradation

to be suppressed

In our preliminary

unlikely

for

and

and antipain

of HU on lysosomal

assays.

HU inhibited

of guanidobenzoic

proteases.

no effect

that

was unexpected

of EGF was reported

lysosomal

amines

was suggested

This

and benzyl

effect

it

effect Whatever

for

study

the

action

of HU on the the

mechanism

of internalized of HU

as

lysosomal

enzyme

would but

turn

out

degradation

an inhibitor

of

by deoxyribonucleosides.

ACKNOWLEDGMENTS This the Ministry

study was supported by a Grant-in Aid for scientific of Education, Science and Culture, Japan. 208

research

from

Vol. 109, No. 1, 1982

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RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. S. 6. 7. 8. 9. 10. 11. 12. 13, 14. 15. 16. 17.

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