Altered nuclear RNA transport associated with carcinogen intoxication in rats

BIOCHEMICAL

Vol. 55, No. 2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ALTERED NUCLEAR RNA TRANSPORT ASSOCIATED WITH CARCINOGEN INTOXICATION Edward

A. Smuckler

IN RATS

and Marlene

Koplitz

Department of Pathology University of Washington Seattle, Washington 98195 Received

September

7,1973

SUMMARY: The release of nuclear restricted RNA to the cytoplasm in dimethylaminoazobenzene (DAR) and acetylaminofluorene (A&?) induced tumors suggested a basis for the phenotypic expression of chemically induced neoplasms. In this report the increased release of RNA from nuclei is identified --in vitro both following acute intoxication and with prolonged (tumor producing) exposure to a third carcinogen, thioacetamide. This release is independent of AT!? added in the cell-free system and consistent with "leaky" nuclei, providing further support for the hypothesis that nuclear RNA transport is involved in chemical carcinogenesis.

The release

to the

other

RNA species,

basis

for

not

benzene

livers

(AR),

the genetic appear

in

liver. the

that

nuclear

information

coding

for

the several

liver provide

been

altered

transport

treated

with

aberration MATERIALS Male

state.

could

be identified

by the

change

In the

for

about

report

the

aminoazo-

that

appearance

also

to test system

but

and neoplastic of

consistent

transport

a cell-free

following

carcinogen

nuclear

We set in

enzymes

in the regenerating

of membrane

(3-9).

(DAR),

RNAs may contain

constitutive

for

of tumors

analog,

These

information

The basis

investigated

carcinogens. produced

genetic

and the presence

differentiated

recently

hypothesis.

and reappear the

(1,2).

the cytoplasm

the noncarcinogenic

this

alpha-fetoprotein

only

with

for

They may also

the less

treated

a basis

developing

in

neoplasia

and by dimethylaminoazobenzene

provided

the

as a potential

induced

RNA appeared

(AAF)

RNA, and of possibly

was suggested

of chemically

restricted

of animals

restricted

manner

expression

by acetylaminofluorene

in the

of nuclear

in an uncontrolled

the phenotypic

The observation induced

cytoplasm

of RNA has whether from

we demonstrate

or not

Dawley

rats

Copyright 0 I9 73 by Academic Bess, Inc. .4N rights of reproduction in any form reserved.

were

two types

thioacetamide.

maintained 499

in

our

laboratories

an

animals

AND METHODS: Sprague

with

on Purina

of

BIOCHEMICAL

Vol. 55, No. 2,1973

Lab Chow in wire thioacetamide stration

bottomed

dissolved

was made by gastric For

long-term

acetamide

for

12 weeks.

hepatic

neoplasms.

sacrifice,

the

animals

were

serum

pH 7.6.

in 0.88

in a total

nuclease

inhibitor

for

by the

addition

centrifuged

volume and the

suspended

presence Total without

data

and in

"zero"

time

injection. were

were

removed,

weighed

(10).

The isolated

assay from

mgm nuclear 8 @i ATP.

for

0.05

of ribo-

Ishikawa

(3).

protein,

37 ug ribowas carried

at 0 and at 20 minutes

the

by centrifugation

suspension

was the

subtracted.

These

500

for

at zero

total results

20 minutes

at 2000 xg for

original

of ATP was determined

RNA release

cold

and

of l/10

1.0 M NaOH, and both

scintillation

This

was aspirated

out in

M Tris

The nuclear

release

and the nuclear

and the

obtained

nuclei

Incubation

The supernatant

in

The

of blood

was stopped

of RNA released

the absence

TCA precipitable the

2.2

was dissolved

the percentage

vein

3 microcuries

samples

adapted

was carried

a liquid

and received

measured.

buffer,

recovered

in

scheduled

as DNA and RNA were

and the precipitate

in Aquas01(~)

before

by tail

thio-

had identifiable

was made 5% TCA by the addition

Precipitation

The precipitate

of the supematant

it

animals

Admini-

0.25%

M MgC12,

15 minutes.

material

given

the same

0.005

The reaction

which

containing

M KCl,

and without

cold

a diet

livers

0.025

a system

ml of ice

following

insoluble

minutes.

these

with

received

ether

prepared

of 0.6 ml,

at 2O'C.

of 2.2

of 50% TCA.

with

as well

volume

at 800 xg for

recentrifuged

the

were

out in

(11,14)

20 minutes

with

to 8.8 mgm/ml of protein,

was carried

contained

fed

concussion,

nuclei

content

was diluted

nucleoprotein

anesthetised

M sucrose

The protein

suspension

out

these

they were

animals

and 120 minutes

determinations,

and from

fast,

85% of surviving

minutes

by cervical

radioactivity

suspended

time

were

100 gram body weight

sacrificed

separately, were

five

per

control

animals

At this

were

C acid

a 16 hour

intubation,

Forty

RESEARCH COMMUNICATIONS

at 5 or 20 mgm/lOO gram body weight.

studies

animals

14

of erotic-6-

Follawing

in water

vehicle.

for

cages.

AND BIOPHYSICAL

nuclei

20

the radioactivity were

measured

spectrophotometer.

From

time,

in the

in

at 20 minutes

the precipitable

radioactivity are shown

material.

released in Table

I.

BIOCHEMICAL

Vol. 55, No. 2, 1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FIGURE 1. (A) An electron micrograph of control nuclei following incubation There is an accentuated chromatin clumping, with ATE' for --in vitro release. the preservation of the nuclear envelope is apparent (arrows). Some small quantity of debris is present (d). Magnification approx. 15,000. (B) h electron micrograph of nuclei following in vitro release from an animal that received 5 mgm/lOO gram body wt. of thioacetamide 16 hours before sacrifice. There is an accumulated clumping of the chromatin, the aggregates are more dense than controls, and the nuclei are larger. Again the nuclear envelope remains and the outer leaflet is present (arrows). Magnification approx. 15,000. (C) Nuclei from an animal 16 hours following 20 mgm/lOO gr body wt. of thioacetamide in vitro RNA release. The chromatin clumping and denseness -of packing is again more notable than control preparation. Nucleolar changes are readily apparent. The nuclear envelope is intact and the outer leaflet is present (arrow). Magnification approx. 20,000. To determine

the size

fluids

following

incubation

placed

on linear

lo-30%

of the

released

and removal sucrose

ribonucleoprotein, of the nuclei

solution

gradients

501

supematant

by centrifugation containing

SO mEI KCl,

were 5 mM

Vol. 55, No. 2,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

502

Vol. 55, No. 2, 1973

I

BIOCHEMICAL

AND BIOPHYSICAL

,100

RESEARCH COMMUNICATIONS

I000

900

700 800

600 700 ,500 t

. 600 d z b 500

z LJ 400

400 300

fr L

3OC 200 200 100

100

c

0 P

0

, 40 w~TOM

IO 20 30 Fraction number

0 TOP

FIGURE 2. Supernatant fluids following sedimentation of nuclei from incubation mixtures were loaded onto lo-30% linear sucrose gradients containing 50 mM KCl, 5 mM MgC12, 50 mM Tris pH 7.6, 0.01 M KHC03. The gradients were centrifuged at 25,000 Rev/min in Spinco SW27 rotors for 14 hours. 45s nuclear material (2,3) was added as marker. The gradients were fractionated and assayed for measurement of radioactivity and O&&j0 as indicated. The major band from control (A), 5 mgm TIAA 16 hours (B), and 20 mgm TIAA 16 hours (C) are identical and migrate at a level with 45s material.

W12,

50 mM Tris

to several

tubes

centrifugation

pH 7.6 with for

comparison RPM for

separated

into

were

fractions

was measured

It possible

ribosomal-like selection

suggested

the separation (120

that

the

of RNA into minutes)

of RNAs might

alter

in

40 fractions,

are shown

(5,9). these

Marker

The released

14 hours

as described,

The gradients

has been

M KHC03.

(3).

at 25,000

gradients

at 260 nm.

0.01

nuclear material

messenger-like

503

detected

The

the several by absorbency

2.

of RNA labeling

To see if

in

were

by

SW27 rotor.

the radioactivity

in Figure

results,

was separated

a Beckman Spinco

and the markers

time

45 S RNA was added

--in vivo

(45 minutes these

manipulations

a separate

set

makes labeling)

and

and possible

of experiments

was

Vol. 55, No. 2,1973

undertaken

using

Nuclei for

BIOCHEMICAL

long-term

before

1 hour

sectioned

The results incubation

dehydrated

pellets

structures

labeling.

and following

at O"C,

were

are shown

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

and embedded

examined

fixed

in

in Epon epoxy

in an AEI-6B

in Figure

are shown in Table

electron

II.

1% osmium tetroxide resin

(12).

microscope.

The These

1.

RESULTS AND DISCUSSION: We were Ishikawa

able

that

dependent,

to confirm

the --in vitro

required

the ribonuclease pellet

before

nuclear

envelope, (Fig.

into

nuclei those

following

nuclei

receiving were

effective

received

able

neither

reflected

in

of

electron

the presence

micrographs

incubation,

there

was an enhanced

zero

time

0 time but

loss

of

of the

of an intact clumping

sucrose

gradient

centrifugation with

longer

--in vivo

were

loss

nor

of

labeling

and an accentuation

changed

of this

504

but

ribonucleoprotein animals

The major

(Table

also

dependent "leakiness," a decreased

complex

released

when measured

by

faster-sedimenting

confirming

change

the 5 mgm

or ATP dependent

an increased loss,

16 and 36 hours. times

and the net

receiving total

(the

these

of the non-ATP

both

and treated

45 S material,

at both

of

80% of controls,

In addition,

Animals

dependent

2).

were

system

a doubling

of the

(Fig.

70%).

a cell-free

dose produced

control

marker

evident

incorporation

the 5 and the 20 mgm doses

I).

showed

The size

comparing

was decreased

(Table

and non-ATP

different

These were

both

ENA in

the higher

transport.

was conincidental

there

was reduced

was not

a persistence

in

the presence

gram body weight

transport,

In contrast

with

product

5 mgm/lOO gram body weight

increased

release.

observed

results

was temperature

revealed

to restrict

ATP transport

ribonucleoprotein

(3-9).

a larger

the

incubation

following

20 mgm/lOO

ATP dependent

release

Furthermore,

intoxication

--in vivo that

less

dose showed

animals

1).

of animals

nuclei

control

nuclear

in

(3,14).

and after

from

assaying

and resulted

and following

By 16 hours orotate

nuclei

system

inhibitor

nuclear

chromatin

ATP,

with

previous Similar

observations

results

11).

In other

was noted

with

species

were experiments

16 week

BIOCHEMICAL

Vol. 55, No. 2, 1973

AND BIOPHYSICAL

TABLE

RESEARCH COMMUNICATIONS

I

PERCENTAGE RELEASE OF --IN VIVO LABELED (45 MINUTES) NUCLEAR RNA TO INCUBATION MEDIA --IN VITRO? Without Added ATP

0 Time 2.00 -+ 0.90

Control

6.82

+- 1.14

10.3

Total Including

With Added ATP

Release 0 Time

+- 2.24

30.00

+- 5.69

32.00

+ 7.40

+- 5.37*

29.95

t- 8.2

32.01

+10.2 -

5 mgm TIAA

16 hr.

2.06

5 mgm TIAA

36 hr.

2.23 -+ 0.88

9.40

+- 4.10*

27.4

t- 6.99

29.6

+ 9.40

20 mgm TIAA

16 hr.

6.39 f_1.56*

15.49

+- 2.99*

30.95

t- 1.79

37.34

-t 4.57

20 mgm TIAA

36 hr.

3.61 + 1.79*

12.10

+- 1.73*

23.86

t- 3.15*

27.47

-t 3.61*

Tumor bearing

liver**

12.1

11.4

27.6

16.2

?Assay for release of RNA from nuclei was measured as described These results represent the means -t one standard deviation (3). separate experiments. *These

values

**Livers

are statistically

from

three

animals

sufficient

to produce

changes

in nuclear

composition,

incubation,

all

three

DNA and 20% of RNA were of the nuclei nucleolar from

was not

changes

is

lost

during

recovered

different,

in

changes

(Fig.

clumping There

1).

prelabeled

DNA.

in nuclei

protein

intact,

apparent

appears

capacity

however,

in nuclei

is no difference

whose

and

The morphology

appeared

Thus it

There

following

pellet.

were

to

fragility.

of the nuclei

envelopes

chromatin

results

in nuclear

the nuclear

nodules.

are unrelated

10% of the original

the nuclear

animals

of tumorous

in

that

loss

--in vivo

to restrict

RNA

in vitro. There

tive

not

thioacetamide

These

about

at P < 0.05.

removal

composition

preparations

or soluble

control

or to differences in the

treated

of TCA precipitable with

following

and accentuated

thioacetamide

treatnmnt

pooled

hepatomas.

difference

in

from

were

feeding

is no significant

different

by Ishikawa -et --al of 3 or more

transport

is

increasing of RNA from

development

evidence

that

the nucleus

and regeneration

the processing, play

(l-9).

505

restriction

a significant The mechanisms

role that

and selecin regulation are involved

BIOCHEMICAL

Vol. 55, No. 2,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE II PERCENTAGE RELEASE OF --IN VIVO LABELED (120 MINUTES) NUCLEAR RNA TO INCUBATION MEDIA --IN VITRO?

0 Time

Without Added ATP

1.40

4.80

28.8

25.2

Control

With Added ATP

Total Including

Release

5 mgm TIAA

16 hr.

1.81

9.10

22.6

24.2

20 mgm TIAA

16 hr.

4.30

12.40

13.4

17.7

?Assay for release (3). The results

in this

modulation

demonstrated studied

of RNA usage

that

in

there

of RNA from nuclei was measured as described represent the mean of two experiments.

transport

a cell-free

function

modify

this

or level

transport

of

this

It

has been

normally

restricted

feeding

regeneration

that

quantities

but

usually

translated

continued

of the phenotypic that

with

*We do not

restricted in

constitutive

reproductive

imply

in

cytoplasmic

factors

that

also

are

differences

the

cytoplasm

carcinogen

for

these

the state

species

506

RNA species

feeding,

but

surface

could

could

Subsequent only represent

transcribed

include

changes

carcinogen messenger

the

and for

be the basis studies

of RNAs

increased

segments

membrane

after

species

The apparent genomic

not

The cytoplasmic

different

and these

Such a change of neoplasia.

of

in

analog.*

liver,

of dimethylsulfoxide, all

recently

development.

enzymes,

expressions

that

More

quantitatively

the adult

that

(9).

after

but

dependent,

(3,4).

RNA may carry

capacity.

the exception

was energy

can be

so isolated

there

appears

and during

nuclear

are

complex

of a non-carcinogenic similar

regeneration

for

there

to the nucleus,

of this

information

there

during

release not

that

--et al.

Ishikawa

described.

transport

in the material

and that

shown

poorly

this

phenomena

of qualitatively during

that

shown

system

equivalent

location occurs

have

only

of RNA as a ribonucleoprotein

system,

was messenger

Webb and coworkers

are

by Ishikawa

for

have feeding RNAs.

some shown is

BIOCHEMICAL

Vol. 55, No. 2, 1973

associated

with

these

associated

with

loss

changes

of restrictional

similar

defect

failure

of ATP dependent

altered

control

are not

that

a membrane

suggests related

--in vitro.

to the process

(13).

In addition, transport. apparent,

AND BlOPHYSlCAL

Our data

indicate

control

--in vivo

with

higher

The mechanism(s) the presence

RESEARCH COMMUNICATIONS

that is

doses

another

associated there

is

underlie

of an intact

nuclear

may be involved.

of neoplastic

transformation

remains

by USPHS grant

CA-13600

How these

with

a

a second

that

alteration

carcinogen

this envelope

changes

are

to be identified.

ACKNOWLEDGEMENT: Cancer

This work Society

was supported grant NP-99C.

in part

and American

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

R. W. Shearer and E. A. Smuckler, Cancer Res. 2, 2104 (1971). R. W. Shearer and E. A. Smuckler, Cancer Res. 2, 339 (1972). K. Ishikawa, C. Kuroda, and K. Ogata, -BBA -3 179 316 (1969). K. Ishikawa, C. Kuroda, and K. Ogata, -BBA -3 213 505 (1970). L. C. Yu, J. Racevskis, and T. E. Webb, Cancer Res. 2, 2314 (1972). T. Kato and M. Kurokawa, Bioch. J. 116, 599 (1970). H. Raskas and S. Bhaduri, Biochem. 12, 920 (1973). E. M. Lukandin, N. A. Aitkhozhina, V. V. Kulguskin and G. P. Georgiev, . FEBS Letters 19, 101 (1971). D. E. Iichumm and T. E. Webb, Cancer Res. 33, 1821 (1973). G. Blobel and V. R. Potter, Science 154, 1662 (1966). A. A. M. Gribnau, J. G. G. Shoenmarkers and H. Bloemedal, Arch. Biochem. Biophys. 130, 48 (1960). This method was modified to use DEAE Sephadex A-25 as the exchange resin. J. Luft, J. Biophys. Biochem. Cytol. 9, 409 (1961). R. W. Shearer, submitted for publication. C. Gagon and G. de Lamirande, Biochem. Biophys. Res. Comm. 51, 580 (1973).

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