Periodic thymidine kinase production in synchronous plasmodia of physarum polycephalum: Inhibition by actinomycin and actidion

Vol. 27, No. 6, 1967

PERIODIC

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

T H Y M I D I N E K I N A S E P R O D U C T I O N IN S Y N C H R O N O U S P L A S M O D I A OF P H Y S A R U M P O L Y C E P H A L U M : I N H I B I T I O N BY A C T I N O M Y C I N A N D A C T I D I O N +)

W.

Sachsenmaier,

D.v.

Fournier,

a n d K.F.

GHrtler

I n s t i t u t e for e x p e r i m e n t a l C a n c e r R e s e a r c h , U n i v e r s i t y of H e i d e l b e r g , G e r m a n y

Received M a y l2, 1967 Mitosis Physarum 26°C.

in m u l t i n u c l e a r

p olycephalum

Nuclear

telophase of the

transferase, interphase senmaier been Hotta

thymidine EC

1965).

1963;

the m e c h a n i s m

of e n z y m e activity suggest

activity

sis of n e w formation

were

increase.

that

enzyme

1966).

The

stages effects

increase protein

1964).

Activity

5'-phosphoat the

of t h y m i d i n e (Bollum

prior

1965;

concerns

studied.

on e n z y m e The

results

reflects

synthe-

in turn d e p e n d s

on the p r i o r

Forscl!ungsgemeinschaft".

655

of R N A

the p e r i o d

(m-RNA).

b y the " D e u t s c h e

of

Actinomy-

synthesis

to a n d d u r i n g

activity

has

Brent

fluctuation

of the a n t i b i o t i c s

of k i n a s e

(Sach-

and P o t t e r , 1 9 5 9 ;

polycephalum.

were

end of

kinase

communication

to i n h i b i t

mitosis

which

following

after mitosis

the p e r i o d i c

in P h y s a r u m

at

3 hrs

and Mueller,

This

employed

of m e s s e n g e r - R N A

+) S u p p o r t e d

first

9 hrs

periodically

of s y s t e m s

on s y n c h r o n o u s the

every

: thymidine

shortly

Stubblefield

at d i f f e r e n t

and

the

Periodicity

controlling

cin C and a c t i d i e n and protein

(ATP

a maximum

Littlefield,

kinase

during

increases

in a v a r i e t y

a n d Stern,

thymidine

synchronously

kinase

2.7.1.21)

a n d Ives,

observed

of the m y x o m y c e t e

e_~tal___u.,1960; S a c h s e n m a i e r ,

and r e a c h e s

et a l . , 1 9 6 5 ; with

occurs

DNA replicates

(Nygaard

enzyme

plasmodia

Vol. 27, No. 6, 1967

METHODS.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNiCATiONS

Disk

polycephalum (Daniel tures

shaped were

cut

between

the

diately

frozen

into

and

National

(control) control

and

mitosis.

maier

et al., and Ives,

of R N A

plasmodial

processed

RESULTS.

segments

average

controls

slowly

half

life

C (Bayer,

with

a Branson

at 2 0 . 0 0 0

(2 ~ c / m l , 0 . 2 5

20 m i n

Synthe-

and were

and Becket,

of a c t i n o m y c i n

decreases

Incorporation

of 3 H - l e u c i n e

over

a period

of s e v e r a l

time

of about

3 hours

the

on the

hours

1964).

C on

of a s y n c h r o -

following

for

of

c/mM)

samples

interphase

of

(Sachsen-

labelling

of 3 H - u r i d i n e

within

soni-

g. P r o t e i n

supernatant.

Labelled

of

the a d d i t i o n

in the

late

piece end

activity

effects

during

third

at the

by p u l s e

Lever-

Chemo-

frozen

(Sachsenmaier

the

imme-

transferred

kinase

5 c/mM).

Incorporation

inhibitor.

drops

3H-uridine

1 demonstrates

20% of the

of the

measured

was

was

The

Cul-

stages

the C a n c e r

20 min.

assayed

earlier

synthesis

by

sonicated

thymidine

was

with

second

3) at 0 ° C f o l l o w i n g

were

as d e s c r i b e d

Fig

were

(2 to 4 ~c/ml,

plasmodium.

below

hand

1965)

and p r o t e i n

nous

and

the

were

1964).

segment

Bethesda).

centrifuged

and p r o t e i n

3H-DL-leucine

RNA

1951)

One

(supplied

earlier

at v a r i o u s

actinomycin

segment

stage

and

mitosis.

Center,

treated

(3 x 1 second,

(Lowry

sis

actidion

as d e s c r i b e d

segments

~g/ml

of P h y s a r u m

and Rusch,

at -30 ° C;

250

Samples

1.0 ml 0.15 M K C 1

equal

stored

Service

the

axenically

third

containing

macroplasmodia

Sachsenmaier

three and

or 50 ~g/ml

therapy

fier

1961;

second

onto m e d i u m kusen)

cultivated

and Rusch,

were

synchronous

rapidly addition

other

suggesting

the b u l k

an

of m - R N A

molecules. Treatment 5 ~g/ml

reduces

20 m i n u t e s

and

with

actidion

protein stops

(Table

synthesis

further

I) at c o n c e n t r a t i o n s

below

synthesis

656

25% d u r i n g completely

the

above first

in c u l t u r e s

Vol. 27, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

I00~ 0/. Ib~ I m

~

&'..

......................

"-.--%

........

e °'o~ .

, |--t

0

!

.

2

.

3

.

fir 4

Fig. I: E f f e c t s of a c t i n o m y c i n C on R N A and p r o t e i n synthesis. 250 gg/ml a c t i n o m y c i n C w e r e a d d e d to the m e d i u m of two p l a s m o d i a 1.6 hrs p r i o r to telophase. At the same 2 time and a f t e r v a r i o u s i n t e r v a l s small s e g m e n t s (2 to 4 cm ) w e r e p u l s e l a b e l l e d on m e d i u m c o n t a i n i n g 2 gc/ml 3 H - u r i d i n e (15 min) or 2 to 4 gc/ml 3 H - l e u c i n e (20 to 30 min) p l u s inh i b i t o r . C o n t r o l p l a s m o d i a w e r e l a b e l l e d s i m u l t a n e o u s l y in the a b s e n c e of inhibitor. E x p e r i m e n t I (o,e); e x p e r i m e n t II (O,I). A b s c i s s a : time f o l l o w i n g the a d d i t i o n of a c t i n o m y c i n C; ordinata: % i n c o r p o r a t i o n of r a d i o a c t i v i t y r e l a t i v e to controls.

TABLE

I: E f f e c t s

Actidion

of a c t i d i o n

Specific after

~g/ml

0 2

5 l0 2O

50

on p r o t e i n

radioactivity 20 and

40 m i n

and R N A

of p r o t e i n

and R N A

incorporation

of

3H-leucine

3H-uridine

dpm (% incorp.) m g prot.

dpm ~g RNA

20 m i n

20 m i n

3820 1540 920 685 920 900

(100) (40) (24) (18) (24) (23)

synthesis.

40 m i n

8 3 2 0 (100) 1990 ( 2 4 ) 1950 ( 2 3 ) 1500 ( 1 8 ) 920 ( l l ) 680 ( 8)

2750 2230 1690 1735 1765 1610

(% incorp.)

(100) (81) (61) (63) (64) (58)

40 m i n

7 1 5 0 (100) 3940 (55) 3760 ( 5 3 ) 3570 ( 5 0 ) 2580 ( 3 6 ) Z530 (35)

P l a s m o d i a l s e g m e n t s w e r e p u l s e l a b e l l e d (20 and 40 min) w i t h 3 H - l e u c i n e or 3 H - u r i d i n e in G 2 - p h a s e (2.5 hrs p r i o r to telophase) i m m e d i a t e l y f o l l o w i n g the a d d i t i o n of the inhibitor.

657

Vol. 27, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

treated with

20 a n d 50

also

RNA

affects

Thymidine approximately m u m at

prior

(fig.

2)

starts

to i n c r e a s e

of m i t o s i s ,

as o b s e r v e d

earlier

1965).

Addition

of 50

the p e r i o d

interrupts

activity does Actinomycin

increase

extent.

and reaches

or i m m e d i a t e l y

tein.

activity

concentrations,

to p r o p h a s e

to or d u r i n g

enzyme

to some

at all

50 m i n p r i o r

the end

a n d Ives,

synthesis

kinase

Actidion,

~g/ml.

increase. due

activity when

to the

expected

onset

of this

longer

inhibited by actinomycin

at a n y

increase This

prevents

increase.

least

Enzyme

C during

the

/2

that

of e n z y m e

only prevents

a d d e d at

time

suggests

to s y n t h e s i s

C on the o t h e r h a n d

of k i n a s e

(Sachsenmaier

actidion

of a c t i v i t y

this

increase

~g/ml

its m a x i -

pro-

the

one h o u r p r i o r induction

last h o u r

is no

of the

i

B I00 -%

Io!

~

8

I

I

,,

o

~

~°'---°--~o ° i 4

~ I/ D / I ^~eI

~ ~ "~LU ~,...

50

~ 0 ~.-/

/._

I o o

,' ,

~.

"J

i I

',

o

,-,

u

~.\v 0

I I

I

-

-2

hr

0

I

-2 hr 0 START OF TREATMENT RELATIVE ro CONTROL TELOPHASE

l

-,

TIME OF ANALYSIS RELATIVE TO TELOPHASE

Fig. 2: E f f e c t s of a c t i n o m y c i n C a n d a c t i d i o n on t h y m i dine kinase induction. A: P o i n t s i n d i c a t e e n z y m e a c t i v i t i e s of c o n t r o l p l a s m o d i a at the time s h o w n on the a b s c i s s a . C y t o l o g i c a l s t a g e of n u c l e i : I ( I n t e r p h a s e ) ; P ( P r o p h a s e ) ; M ( M e t a p h a s e ) ; A ( A n a p h a s e ) ; R ( R e c o n s t r u c t i o n of the n u c l e o lus). B: T h e a b s c i s s a i n d i c a t e s the time w h e n 250 ~ g / m l a c t i n o m y c i n C or 50 ~ g / m l a c t i d i o n w e r e added. A l l c u l t u r e s w e r e a n a l y z e d w i t h i n a p e r i o d of 30 m i n a f t e r c o n t r o l telephase. A c t i v i t y is e x p r e s s e d as % of the m e a n of c o n t r o l values.

658

Vol. 27, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

"actidion

sensitive"

thymidine

kinase

period.

is f o r m e d

This

about

suggests one h o u r

that m - R N A prior

for

to e n z y m e

synthesis. The closely

effects parallel

t i o n of the onset

or d e l a y s

indicates

onset

and

TABLE

of this

effects prior

further have

that

kinase

enzyme

II.

the

progress

been

(hrs

are

relative

prevents (Table

the

II).

et al.

required

formed

Addi-

Simi-

(1965).

for

during

the

the p e r i o d

C which

added

t h a n 90 m i n p r i o r

later

C and actidion

stage

no

longer

on m i t o s i s .

of n u c l e i

of t r e a t m e n t

End (0.5 h r s

to t e l o p h a s e )

on m i t o s i s .

by Cummins

proteins

induction

Actinomycin

of a c t i n o m y c i n

Cytological Start

some

when

agent

prophase

reported

induction.

kinase

of m i t o s i s

of m i t o s i s

induction

Effects

to late

at l e a s t

for p r o g r e s s

of t h y m i d i n e inhibits

on t h y m i d i n e

inhibitor

lar o b s e r v a t i o n s This

of a e t i d i o n

of t r e a t m e n t

after

control

Control

Actinomycin

telophase) Actidion

-2.4

I

R

I

I

7

I

R

IP+

I°

-1.4

I

R

IP+

I°

-0.6

IP

R

M

IP °

-0.4

IP

R

R

M

-i.

-0.3

P

R

R

M

-0.2

M

R

R

R

P l a s m o d i a l s e g m e n t s w e r e t r a n s f e r r e d onto m e d i u m c o n t a i n i n g 250 ~g/ml a c t i n o m y c i n C or 50 ~g/ml a c t i d i o n at the time indic a t e d in c o l u m n (1). T h e c y t o l o g i c a l s t a g e of n u c l e i w a s o b s e r v e d at the s t a r t of t r e a t m e n t ( c o l u m n 2) a n d 0 . 5 h r s a f t e r c o n t r o l t e l o p h a s e ( c o l u m n s 4 + 5). I: I n t e r p h a s e ; IP: E a r l y p r o p h a s e ; P: P r o p h a s e ; M: M e t a p h a s e ; R: R e c o n s t r u c t i o n of n u c l e o l a r m a t e r i a l (0.2 to 1.5 hrs a f t e r t e l o p h a s e ) . (+,o): a b n o r m a l l y c o n d e n s e d c h r o m a t i n a n d s m a l l (+) or n o r m a l ( O) n u c l e o l i .

659

Vol. 27, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

to telophase,

still

35 m i n b e f o r e

telophase.

cules

required

or f u n c t i o n Our

trolled

a pulse (Jacob

prior

and Monod,

1961);

turnover

by

at

end

kinase

b)

This

thymidine

of the

cycle

con-

follow-

synthesis

kinase

level;

as a

m a y be

of the

a)

enzyme

later,

m-RNA.

of

cistron

or a c t i v a t i o n

translation

stabilization

one

mitosis:

mole-

activity

process

triggers

as

formed

specific

kinase

stabilization

the

are

of the d i v i s i o n

which

at the

as late

that m - R N A

of m i t o s i s

synchronous

molecules

added

indicate

induction.

mechanism

m-RNA

when

thymidine

the

of m - R N A

kinase

steps

that

at

to each

may

thymidine

enzyme

by a c l o c k

thymidine enzyme

suggest

of p e r i o d i c

ing e v e n t s

than

periodically

mitosis

This

certain

shorter,

results

increases result

for

affects

of

c) b l o c k

of

molecule.

REFERENCES

Bollum,

F.J.,

and Potter,

V.R.,

Cancer

Res.

1_99, 561

(1959).

Brent, T.P.; Butler, J.A.V.; and C r a t h o r n , A.R.; N a t u r e 207, 176 (1965). Cummins, J.E.; Brewer, E.; and Rusch, HoP.; J. Cell Biol.

2_Z, 337 (1965) Daniel, J.W., and Rusch, H.P., J. Gen. M i c r o b i o l . 25, 47 (1961). Hotta, Y., and Stern, H., Proc. Natl. Acad. Sci. 49~, 648 (1963). Jacob, F., and Monod, J°, J° Molec. Biol. 3, 318 ~ 9 6 1 ) . L i t t l e f i e l d , J.1~., B i o e h e m . B i o p h y s . A c t a T14, 398 (1966). Lowry, 0.H.; R o s e b r o u g h , N.J.; Farr, A.L.; and Randall, R.J.; J. Biol. Chem. 193, 265 (1951). N y g a a r d , 0.F.; Guttes, S.; and Rusch, H.P.; B i o c h e m . Biophys. A c t a 38, 298 (1960). S a c h s e n m a i e r , I¢., B i o c h e m . Z. 34Q, 541 (1964). S a c h s e n m a i e r , Wo , and Rusch, H.P., Exp. Cell Res. 36, 124(1964~ S a c h s e n m a i e r , ~., and Becket, J.E., Mh. Chem. 94, ~ 4 (1965). S a c h s e n m a i e r , W., and Ires, D.H., Biochem. Z. $-~33, 399 (1965). S t u b b l e f i e l d , E., and M u e l l e r , G.C., Biochem. Biophys. Res. Commun. 2__O0, 535 (1965).

660