Effects of 1-methyl cyclohexane carboxylic acid (CCA) on cellular energetics in neuroblastoma cells

Vol. 103, No. 3,198l December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1044-1051

15, 1981

EFFECTS OF I-METHYL

CYCLOHEXANE CARBOXYLIC ACID (CCA)

ON CELLULAR ENERGETICS IN NEUROBLASTOMA CELLS Bernard Croizat, Francis Berthelot, Marie-Madeleine Portier, Hdlbne Ohayon* and Franqois

Gros

Laboratoire de Biochimie Cellulaire, CollQe de France, 75231 Paris Cedex 05, France, et *Service de Microscopic Electronique, Institut Pasteur, 75015 Paris, France

Received

October

30,1981

SUMMARY The level of cellular energetics has been estimated in neuroblastoma cells under dif erent culture conditions. The cellular accumulation of isomerase-2-deoxyp4 Cf D- glucose-6-phosphate was taken as reflecting glucose utilization. A considerably higher amount of radioactivity - 2.5 to 4 times - is found in CCA treated cells, as compared to other types of cultures, corresponding to a higher rate of deoxyglucose penetration and utilization. INTRODUCTION Cloned possess

cell

lines

many neuronal

culture,

the

properties

cells

neuronal

model

exhibit

for

since

its

injection

studying It

and anticonvulsive, differentiation proteins

cells in

dimensional stimulated

and methionine

expression

(4).

and control

to check,

(6). incorporation

that

by neurite

Synthesis into

were

[I-14C]D-glucose-6-phosphate

complex

0006-291X/81/231044-08$01.00/0 Copyright All righa

0

I981

by Academic Press, in any form

of reproducfion

Inc.

reserved.

acid

some insoluble 2-deoxy

(CCA),

antianoxic

CCA promotes

compared

the

vitro,

i.e.

extension

to be

terminal

carboxylic

of vimentin

; 2DG-6-P,

of

considered

in

effects,

We showed

cultures

are during

cyclohexane

as evidenced

electrophoregrams

they

has pharmacological

conditions

and biochemical

reason,

interesting

of 1 methyl

on the brain

CCA-treated

appropriate

physiological

For this

was thus

mouse neuroblastoma

Under

the genetic

the rat

of the cells from

(l-3).

neurone.

on neuroblastoma

the C-1300

morphological,

differentiation.

effect

from

characteristics

of the mature

a convenient

derived

a marked (5).

The

on two-

was strongly proteins, D-glucose-6-phos-

which

BIOCHEMICAL

Vol. 103, No. 3,198l

are presumably

membrane

was increased.

The synthesis

However,

molecular

course

these

effects

within

the cell.

effects

of anoxia

more generally, purpose,

by Sokoloff

(7,

initially second

the

complex

phosphate activity

reaction between isomerase

blastoma MATERIALS

cell

was not

possible

within

seemed that

cultures

it

rat might

brain

sections.

glucose

but

with here

brain

with

in

This Labelled

the biochemical

oxygen

culture

uptake

or, For that

complex

was used earlier

deoxyglucose

is

does not proceed second

beyond

reaction

is

and the hexose

the amount

with

the

cultures.

can be considered obtained

we used

against

viva

approach

of this

Consequently,

to other

the

[14C]2DG-6-P

IJ4C 3 deoxyglucose

the results

as compared

to correlate

influence

the process

the proteins

to the

such a correlation,

The product

accumulates.

modified.

related

of neuroblastoma

utilisstion.

phosphorylated

which

We report

to find

of the isomerase-_

of the pathway. the

the brain

energetics

rat like

co-precipitating activity.

it

of glucose

8) with

metabolized

the

been clearly

accumulation

as a measure

was also

not yet

CCA protects

the cellular

the cellular

was taken

it

(4),

the cytoskeleton,

have

the drug

Since

with

and isotubulins

In an attempt

approach.

RESEARCH COMMUNICATIONS

associated

of actin

Moreover, of

BIOPHYSICAL

proteins

events

effects

a more direct

cell

bound

of neurogenesis.

pharmacological

AND

of radioas a measure

CCA-treated

of

neuro-

conditions.

AND METHODS

Cell culture. We used the NIE-115 clone from mouse neuroblastoma C-1300. The conditions of culture have been previously described (9). The cells were grown attached to a Falcon culture dish. They extend neurites when the serum is withdrawn from the medium or when CCA or Me2SO is added to the serum-containing medium. Cells kept in the serum-containing medium in the absence of drug were either grown in logarithmic phase or maintained at confluence in a stationary phase. I mg/ ml order to (4). The incubation every 24

Drug treatment. CCA-----------T----treated cultures. The CCA concentration was -of medium (6 x lo-3 M). This high concentration was selected in correspond to those previously used in pharmacological studies cells were maintained for three days in these conditions prior to with [14C]deoxyglucose. Cultures were changed with fresh medium hours.

Me2SO treated cultures. Cultures were kept for seven days in -- :-:---------------3 medium contarning 2 % Me2SO prror to labeling in the same medium. The medium was changed every 24 hours.

1045

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t (min.)

FIG. 1. Cellular incorporation of [14C]2-deoxyglucose. Total radioactivity was measured in the 12 000 8 supernatant and normalised to 106 cells. Cells Each value is the average of four were counted with a haemocytometer. successive countings. M CCA treated cells ; serum free cultures ; Me2SO treated cells ;A ---A growing cells in logarithmic phase ; A---I confluent cells in stationary phase.

[14C]deoxyglucose labeling and samples preparation and treatment. Media were renewed 30 minutes prior to the addition of 1 uCi/ml of 2-deoxv [l-14C]D-glucose (57 mCi / mmole) (Amersham). The 10 cm dishes were inocul-ated so that they contained about 2.5 x 106 cells at the time of deoxyglucose incorporation for every type of culture except for confluent cells in stationary phase which contained 2 x lo7 cells. Deoxyglucose incorporation ranged from 10 minutes to 90 minutes. The dishes were then placed on ice, rinsed with cold saline buffer : Tris-HCl 10s2 M pH 7.6, NaCl 0.25 M, MgC12 10-2Mand lysed with Nonidet P-40. After centrifugation for 10 minutes at 12 000 8, 200 1.11 samples of the supernatant were collected : i) some samples were directly counted in Bray scintillation mixture to account for total cell radioactivity ; ii) other samples were added with 1.8 ml ethanol. After 10 minutes at 0°C the ethanol precipitate was spun down for 20 minutes at 16 000 &. The pellet was resuspended in 0.5 ml Tris/NaCl/Mg buffer and counted in 5 ml Bray scintillation mixture to account for the radioactivity bound to proteins. 2DG and 2DG-6-P are not ethanol precipitable. Consequently the radioactivity in the precipita e corresponds to the 14C actually bound t,o protein, i.e. to the isomerasecomplex. E. 14C 3 2DG-6-P RESULTS Two cells

in

types

logarithmic

[14,]2DG

of

non-differentiated phase,

uptake.

ii)

Fig.

cultures non-dividing

1 shows

1046

cells

the

kinetics

were

employed

kept

of

in

stationary

2-deoxyglucose

: i)

growing phase.

BIOCHEMICAL

Vol. 103, No. 3,198l

AND

0

FIG. 2. Measurement ated to A---A

as measured

more rapid tions.

precipitated

kinetics

After

growing

cells versus

corresponds

t(min.)

radioactivity.

penetration,

and twice

Ethanol

as high

those

precipit-

the amount

as in confluent

from

is very

2DG-6-P

to account its

for

the

phosphorylation

cells

three

shown

on fig.

was also

found

in

cells

and its

1047

was and in

(48 000 cpm per

IO6

14 C radioactivity

isomerase-2DG-6-P

complex.

the activity

of glucose

Total

radioactivity

may be

binding formation.

2. A considerably

the CCA-treated

free

condi-

since

successive

p4 CI 2DG- 6-P complex

are

(8).

the other

in serum

times

markedly

of radioactivity

: 2DG, 2DG-6-P,

low in nervous

with

and total

may be neglected

various

expressed

obtained

in Me2S0 treated,

molecules

after

to CCA cultures

that

than

radioactivity

15 000 and 25 000 respectively),

Isomeraseprecipitation

corresponding

in CCA cells

formed

assumed

cell

of incubation,

to different

phosphatase

total

of 2DG uptake

90 minutes

higher

Products

by counting

The curve

3 times

fraction

of ethanol

loo

COMMUNICATIONS

from the 12 000 8 supernatant was measured and normalised 106 cells. CCA treated cells. o-----d serum free cultures ; He2SO treated cells ; a---Agrowing cells in logarithmic phase ; confluent cells in stationary phase.

of incubation.

then

50

RESEARCH

radioactivity

uptake,

cells

BIOPHYSICAL

cells.

metabolic

steps

i.e.

2DG

to isomerase. The results higher

uptake

The yield

of ethanol into

of ethanol

this

BIOCHEMICAL

Vol. 103, No. 3,1981

AND

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RESEARCH

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TABLE I. Percent of the cellular [14C]2DG recovered the isomerase-p4C]2DG-6-P complex

IO min

60 min

45.9

50.3

cells

30.2

37

cultures

18.6

37.3

27.7

40.3

36.9

44.4

CCA treated

cells

Me2SO treated Serum free Growing

cells

Confluent

as

(log.

cells

phase)

(stat.

phase)

These percentages were calculated by dividing the values from fig. 2 (c.p.m. in the ethanol precipitate from 12 000 g supernatant) by the corresponding values from fig. 1 (c.p.m. in the 12 000 4 aupernatant). See RESULTS.

precipitation

is

less

considered

as minimal

of precipitation

total

radioactivity

table

I indicate

ly higher

comparable

for

that,

shown

it

in fig.

culture

time

studied,

cells.

That

means that

independent

of the effect

the yield so that

radioactivity

populations.

at every

that

conditions,

of ethanol-precipitated cell

2 must be

may be assumed

the various

in the different

in CCA treated

the cell

Nevertheless,

the percentages

by CCA treatment, through

100 % and the values

values.

is

one may compare

than

this

versus

The results percentage

complex

is

shown in significant-

formation

is

enhanced

of CCA on the penetration

of 2DG

membrane.

DISCUSSION In this designed

by Sokoloff

various

physiological

markedly

increased

CCA-treated cell

shown)

cells

energetics.

number,

study

but were

neuronal

to evaluate

accumulation ; this

expressed

we have used

cerebral

The main

activity

result [

expressed

as a function

similar.

1048

here

here

14 C 2DG-6-P 1

may be assumed

are

the elegant

method

in response

reported

of the isomerase-

accumulation

The 14 C counts

cells,

local

conditions.

the results very

with

to

concerns complex

in the

to reflect

the level

as a function

of cell

of protein

content

the

(data

of

not

BIOCHEMICAL

Vol. 103, No. 3,198l

Many variables We have paid

specific

ii)

cell

effects

Both

attention replication,

to obtain

and CCA-treated these

agents

Measurements

were

i.e.

cells

allows

morphogenesis,

also

inducers.

induction

made in

in logarithmic

phase

and iiii) were

in altered

added

to occur

two types

culture

by

trophic

condi-

cultures.

in a 7.5 % fetal in the absence

calf

of inducers.

of non-differentiated and confluent

the

induced

to Me2 SO treated

were

growth

energetics.

trophic

Cultures

morphogenesis compared

COMMUNICATIONS

of cellular

neuronal

were

normal

levels

of i)

neuronal

cultures

RESEARCH

to the effects iii)

of neuronal

serum medium which

growing

to the

of the differentiation

serum deprivation tions,

BIOPHYSICAL

may contribute

a particular

conditions,

AND

cultures,

cells

in a stationary

phase. The level

of cell

and the accumulation P igher

cells

stationary

to promote to its

found

between

a complete

stimulation

medium.

the "confluent

These

deoxyglucose

cells"

correspond

(10,

increases

in the oxygen

in neuroblastoma deprivation measurements approach. by classical

cells

as compared may reveal Morphological agentshas

14

[

is

in confluent

level.

CCA appears

cells

Thus,

from glucose

to a different

penetr-

difference and cells

the effect

cells

3 to 4 times with

No significant

emphasize

CI 2DG uptake

of cultures,

pathway

cellular

was

growing

in

due to CCA. The

may reflect

of Walum et al. who observed

cells"

In contrast

types

serum deprived

observations

the

complex,

an intermediate

cells,

observed

uptake

other

of the metabolic

situation

phase

in all

to D-fructose-6-P.

Me2S0 treated

from

1y4CeI 2DG-6-P

reaching

isomerisation

intermediate

"stationary

than

cells

a global

ation

estimated

of the isomerase-

in CCA-treated

confluent

energetics,

the fact

state

from

a decrease

that

the

in the

11).

to our

results,

Nissen

uptake,measured that

minor

grown

changes

been

reported

to differentiate

in a complete that

differentiation also

(12)

by a micromanometric

had been induced

to cells

et al.

are not measured

induced reported

1049

medium

small

technique, by serum (13). with

These our

by serum deprivation

to be accompanied

by minor

or changes

BIOCHEMICAL

Vol. 103, No. 3,198l

at

the level

of mRNAs and proteins It

metabolism

is

significantly

is

drastically

synthesis

ation).

classical

that

membrane

effects

that

Several

that

neuroblastoma

and that

a fundamental

role

in

are not neuronal

the burst triggering

not

shown).

bound

CCA also

after usually

CCA-treated

energetics

cells

to oligomycin stimulates

the

and cytoskeleton addition screened

by CCA (manuscript

a more complete

the neuronal

in the ultra-

resistance

proteins

observed

in cell

in neuroblastoma

For example,

and their

induced

CCA promotes

changes

by modifications

markers,

are markedly

agents,

We believe

drastic

microscopy.

(data

RESEARCH COMMUNICATIONS

14, 15).

more mitochondria

serum deprivation. with

by electron

increased

(6),

(9,

to note

of some insoluble

components

BIOPHYSICAL

by CCA are accompanied

as observed

contain also

interesting

induced

structure

AND

neuronal reported

of Me2 SO or in studies in preparexpression here

in

may play

differentiation.

ACKNOWLEDGMENTS This work Fransoise Perrot for typing the manuscript.

has been supported by a grant from SANOFI. We thank her excellent technical assistance and Denise Baron

for

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11. Walum, E. and Edstrgm, A. (1976) Exptl. Cell Res. 97, 15-22. 12. Nissen, C., Ciesielski-Treska, J., Hertz, L. and Mandel, P. (1973) J. Neurochem. 0, 1029-1035. 13. Hertz, L. (1966) J. Neurochem. 13, 1373-1387. 14. Felsani, A., Berthelot, F., Gros, F. and Croizat, B. (1978) Eur. J.. Biochem. 92, 569-577. 15. Grouse, L.D., Schrier, B.K., Letendre, C.H., Zubairi, M.Y. and Nelson, P.G. (1980) J. Biol. Chem. -255, 3871-3877.

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