Studies on the regulation of ornithine decar☐ylase activity by the microtubules: The effect of colchicine and vinblastine

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Vol. 68, No. 2, 1976

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STUDIES ON THE REGULATION OF ORNITHINE DECARBOXYLASE ACTIVITY THE EFFECT OF COLCHICINE AND VINBLASTINE

THE MICROTUBULES:

Kuangyu

Chen, John Heller,

Department

November

and E. S. Canellakis

of Pharmacology,

New Haven, Received

BY

Yale

Connecticut

University 06510

3,1975

SNmARY : Colchicine and vinblastine in micromolar concentrations inhibit the activity of ornithine decarboxylase (E.C.4.1.1.17) (ODC), of mouse leukemia L1210 cells, which has been stimulated by dilution of the cells with fresh medium and serum. The colchicine analogues, lumicolchicine and colchiceine, which do not affect microtubular strcuture, do not inhibit ODC activity even of the microtubular structure at 10s4E4. However, it appears that disruption is not in itself enough to inhibit ODC activity but that one or more additional temperature dependent steps are involved. We propose that the microtubule system is one of a series of components which regulates ODC activity. Based

on the study

by ConA, dimeric workers are

(1,2)

mediated

succinyl proposed

system

should

in general

Recent

work

mosaic

separation

bition

by colchicine also

high

tion

cells of fresh

reagents

that

and puromycin

sites

and his

most

of erythrocyte

ghosts

stimulation

likely

the

of the microtubule by the

sites

co-

interactions

mediated

and transport

the

high (6).

transferred

rate-limiting

levels

(4)

cell

surface.

(3),

the

and the inhi-

of lymphocytes

ODC activity from

medium

and serum

affect

cell

growth,

is

the resting (7).

It

in

increased to the

can also

such as cyclic

(10).

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

enzyme in

of activity

401 Copyright All rights

mobility

by ConA

hypothesis.

exhibits

are

the disruption

and glycophorin

decarboxylase,

of growth

proteins,

the information

of the mitogenic this

pathway,

tured

abolish

Edelman

receptor-cytoplasmic

hypothesis,

of phagocytic

support

rates

to this

receptor

drugs,

of colchicine-binding

on the spectrin

Ornithine thetic

the membrane

by an assembly According

of lymphocyte

ConA and anti-mitotic that

microtubules.

(5)

of the modulation

tissues

the polyamine or cells

dramatically growing

be induced AMP (8),

state in vivo

biosynwith

when culby the addiby various

dexamethasone

(9)

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Vol. 68, No. 2, 1976

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In previous experiments, we have shown that antibodies to L1210 membrane proteins in the absence of complement will ably by cell surface interactions

(11,12).

experiments from this laboratory

indicated

associated with a decrease in ODCactivity

inhibit

the growth of cells;

Furthermore, preliminary that this inhibition of L1210 cells.

presum-

unpublished

of growth was

Consequently, it

seemedreasonable to speculate that these various responses of ODCcould be associated with perturbation In testing inhibit

a variety

ODCactivity

of the plasma membranes.

of microtubule disrupting

agents we found that they

which has been stimulated by dilution

with fresh mediumplus serum. In contrast,

structural

of the L1210 cells

analogs which are known

not to affect

the microtubule system, such as lumicolchicine

had no effect

on ODC activity.

and colchiceine

MATERIALSANDMETHODS Chemicals. Colchicine , pyridoxal phosphate were purchased from Sigma Chemical Company, St. Louis, MO. DL-[1-14C]ornithine (sp. act. 7.5 mCi/mmole) and aquasol were products of NewEngland Nuclear; DL ornithine monohydrochloride was obtained from Nutritional Biochem. Corp. Vinblastine was obtained from Eli Lilly Co. and cytochalasin B was from Aldrich. NCS tissue solubilizer was purchased from Amersham/Searle Corp. Lumicolchicine and colchiceine were kindly given by Dr. G.T. Shiau, Department of Pharmacology, Yale University. Cell culture. L1210 cells were grown as d&scribed previously (13). The cells were grown to the late log phase (1.5 x 10 /ml) and hen diluted with fresh Fisher's mediumplus 10%horse serum to about 4 x 10I /ml for all subsequent studies. Assay of ODCactivity. Approximately 6 x lo6 cells were harvested by centrifugation and washed twice with PBS at 4'C. The cells were resuspended in 0.4 ml 50 mMTris (pH 7.2 at 37'C) containing 0.1 mixEDTA, 0.05 mMpyridoxal phosphate, 10 mMdithiothreitol and disrupted by one cycle of freezing and thawing. Enzymatic activity was assayed according to Fong et al. (14) with the following modification. Cell homogenate 0.1 ml and 10 1-11ornithine(0.05 umole, 7.55 nCi/nmole) were put into a 17x100 mmplastic tube with a well containing 0.1 ml NCS-toluene (1:l V/V) suspended from the cap. The reaction was stopped with 0.2 ml of 10%TCA after 60 minutes at 37'C and the tube was incubated for another 90 minutes to insure complete absorption of the 14C02. The radioactivity was determined by placing the well into 10 ml of aquasol and assayed in a Packard Tri-carb liquid scintillation spectrometer. RESULTSANDDISCUSSION 1.

Time course of stimulation Following dilution

of ODCactivity

of stationary

(Fig. 1).

L1210 cells,

402

an increase in ODCactivity

BIOCHEMICAL

Vol. 68, No. 2, 1976

AND BIOPHYSICAL

0

Fig. 1.

peak activity phase prior

to dilution

of the cells.

2-6 hours.

in the control 10W6Mvinblastine

of ODCactivity.

colchicine

at the stationary

as described in Fig. 1. at the time of dilution.

vinblastine,

of ODCactivity

Vinblastine

system (1s).

on the microtubular

colchiceine

Lumicolchicine,

were added of ODCactivity

at 7 x 10m7M. This is

of vinblastine

in disrupting

an isomer of colchicine

does not inhibit

403

stimulation

concentrations

caused a 50% inhibition

system (lo),

and lumi-

(Fig. 2).

produces a 50% inhibition

in agreement with the greater effectiveness

this

effect.

was assayed 1 hour after

Drugs at the appropriate

at 8 x 10~'I.! whereas colchicine

dilution

added at time zero completely inhibits

Colchicine has a similar

on the inhibition

In Fig. 1,

sample is attained 2 hours after

In this experiment enzyme activity

effect

The exact time at which the

(K-Y. Chen, unpublished observations).

Dose-response curve for colchicine,

microtubular

V inblostine

is attained depends on the cell concentration

the peak activity

2.

x lo+

Time course of the effect of vinblastine on the stimulation of ODC activity. Stationary L1210 cells were diluted with Fisher's medium plus 10%horse serum in the absence (0) or in the presence (0) of 5x10T6M vinblastine. Cells were collected at the designated times and ODCactivity was measured as described in the text.

occurs which reaches a maximumafter

stimulation

Control

. +5

3or

RESEARCH COMMUNICATIONS

ODCactivity

the

which has no even at

Vol. 68, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

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o Colchicine l

Vinblostine

0 Lumicolchicine A Colchiceine

01-e IO

Fig. 2.

5 x lo+.

IO IO Molarity

IO

to

Dose-response curve for colchicine (o), vinblastine (o), colchiceine (A) and lumicolchicine (p) on the inhibition of ODCactivity of L1210 cells. Cells were diluted and various drugs were added simultaneously ODCactivity was assayed 1 hour after at different concentrations. dilution.

Colchiceine,

as an anti-mitotic

an analogue of colchicine

drug (17), inhibits

these or higher concentrations,

which acts much less effectively -5 ODCactivity only 20% at 10 M. At

none of the compoundsaffect

ODCactivity

when

added to the enzyme assay, in vitro. 3.

The effect

of cytochalasin

of L1210 cells

B and ouabain on the stimulation

(Fig. 3).

Since it has been proposed that the microtubular tively

with the microfilament

microfilament activity

of ODCactivity

specific

system functions

system as the cytoskeleton

reagent, was tested.

(2) cytochalasin

We found that it inhibited

by 50%at 7 x 10m6M. We also tested ouabain, an inhibitor

ATPase, and found only 20%inhibition

of ODCactivity

these compoundsaffected

when added directly

ODCactivity

404

cooperaB, a ODC

of (Na++K+)-

at 10m5M. Neither of to the enzyme assay.

Vol.

68,

BIOCHEMICAL

No. 2, 1976

AND

I

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A Ouabain

01

lo-

IO

to

IO -4

lo-5

Molarity

Fig.

3.

Dose-response curve for hibition of ODC activity tochalasin B or ouabain trations. ODC activity

Temperature

1.

Various is

effect

15 minutes

to ask the question the inhibition

tivity tions

of cells

increased cells/hr whether

OK

(15,19).

whether

We took

for

incubated

the presence

activity.

while colchicine

of the microtubule

the

cold

is

disrupted

advantage

of this

experiment of L1210

60 minutes

We found

both

that

of control

cells

is

not

enough

had a value This in

405

cells,

in order

in itself

that

to inhibit

por-

the presence

not

enoug}

ODC ac-

and then

and

treatment

alter

the

already

was 754 cpm/105

of 740 cpm/105

indicates itself

is

low temperature

The ODC activity

or not.

of cells

observation

in

did

cells

storage

the cellular

of colchicine

treated

system

of the microtubules

1 hr by dilution at 0°C for

microtubule after

or absence

was present system

it

For this

of 10-6 bl colchicine. in

the cytoplasmic

the disruption

stimulated were

in the absence of the cells

and that

of ODC activity.

had been

of L1210 cells.

have shown that

to low temperature

at 0' C for

for

on ODC activity

investigations

sensitive

cytochalasin B (0) and ouabain (A) on the inof L1210 cells. Cells were diluted and cywas added simultaneously at different concenwas assayed 1 hour after dilution.

the ODC

cells/hr disruption activity

and

BIOCHEMICAL

Vol. 68, No. 2, 1976

that

there

must exist

microtubules

also

inhibit

inhibit

the uptake

inhibit

many secretory system Although

reaction their

of these

process range

should

drugs drugs

with

binding

these

given

nucleoside

generally

are

system

(24).

cells

but

acid

(20).

do not

(21).

They

considered

to be micro-

processes,

the

not

process

are not

be osbserved

should

a process

which

that

level

is

biological

such

10

is

depends

M or

temperature

processes

the effect

-6

as lumicolchicine

A good example

dependent

who showed

known

be around

inhibition

at O'C.

only

1) The concentration

anti-mitotic

microtubule (21)

dependent

(25).

3) The colchicine

should

is

this

which

on the molecular

A microtubule criteria

inhibit

process.

and Wilson

transport

of applying from

others

of colchicine

on

on the integrity

of the

system.

of ODC activity

inhibits

it

B at 10-4M

Mg2+ -ATPase,

some plasma

of ODC activity

by colchicine

enzymes

colchicine

Furthermore,

ouabain

which

ODC activity

even at 10m4M.

etc. is

suggest

colchicine,

membrane (26).

It

primarily

due to the

inhibits

(Na++K+)-ATPase

406

vinblastine

seems unlikely

ODC activity

may also

interactions

enzymes activities,

inhibits

regulation

the microfilament system

receptor-cytoplasmic

the

of the cytoplasmic

to affect

the microfilament

that

that

to the integrity

is thought

that

reported

strongly

related

of membrane

adenylcyclase,

because

data

B, which

has been affect

is

indicating

the mediation

Recently

our

cells

Cytochalasin ODC activity

in

criteria

of L1210

microtubules.

sin

tubulin

drugs

Based on the above

volved

of the

drugs

isobutyric

many biological

and differentiating

microtubule

o-amino

by the following

this

by Mizel

in mammalian

the biological

with

no inhibition

criteria

or which

analogues

inhibit

dependent,

transport

may affect

anti-mitotic

not

the disruption

(22,23).

2) Colchicine

lower.

is

processes

can be defined where

between

are known as anti-mitotic

of 2-deoxyglucose

these

specific

alkaloids

nucleoside

related

steps

of ODC activity.

and the vinca

drugs

tubule

dependent

and the inhibition

Colchicine These

temperature

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

system, be in-

(2).

and cytochalaQ.(Na++K+)-ATPase,

that

inhibition

at much lower does not

the inhibition of these

membrane

concentrations. significantly

inhibit

Vol. 68, No. 2, 1976

BIOCHEMICAL

Our low temperature bule

system

itself

that

additional

does not

Although from

cytoplasm

its

or nucleus,

disrupting

agents

the membrane particular occur

surface

with interest

between

we do not

receptors

provides

a model

a cytoplastic because

are

involved

know exactly

that

of the

which

between

which

the

assumed disruption

in ODC activity.

system is

connects

marker.

interaction

we have

how the cell

ODC activity

system

of the microtu-

therefore,

decrease

enzymatic

and tubulin

disruption

to the microtubule

the discovery

spermine

steps

and the observed

stage

the

ODC activity,

sensitive

system

at this

shows that

inhibit

temperature

of the microtubule

mation

experiment

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

These

transfers

and then

affected

infor-

to the

by microtubule

the perturbation results

has been

also

of acquire

demonstrated

to

(27,26).

Acknowledgment: The authors wish to thank Ms. S. M. Tu for her excellent technical assistance. We are also grateful to Dr. G. T. Shiau for the helnful discussions. This work was supiorted by American Cancer Society GranE'BC75 and USPHS Grant CA-04S23 and a USPHS Research Career Award to E.S.C. No. K06-GM-03070.

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