A cytoplasmic membrane-associated DNA from rapidly proliferating tissue

BIOCHEMICAL

Vol.54,No.2,1973

AND BIOPHYSICAL

A CYTDPLASMIC MEMBRANE-ASSOCIATED Bill Departments

DNA FROM RAPIDLY PROLIFERATING

Novak and Howard

of Medicine

Received

July

31,

TISSUE *

Elford

and Physiology

Duke Medical Durham,

RESEARCH COMMUNICATIONS

and Pharmacology

Center

North

Caroline

27710

1973

Summary. A membrane fraction obtained from the post-microsomal supernatant of Novikoff hepatoma and rat tissues has been shown to contain nucleic acid which can stimulate DNA synthesis. Based on the insensitivity to specific RNases and the sensitivity to DNase, the stimulatory nucleic acid is presumed to be DNA. The occurence of this cytoplasmic membraneassociated DNA is related to the proliferative state of the tissue. Since

the work of Bollum

of DNA polymerase wide

variety

bution

et al --

supernatant

tains

levels

It fraction

synthetase weight

of a high

remains

demonstrated

(9) site

obtained

that for

(lo)],

studies from

of Baril

the postrat

liver

con-

S) DNA polymerase. this

cytoplasmic

several

reductase,

template

but may have phy-

Recent

(6-8

in a distri-

of its

and regenerating

weight

and dCMP deaminase

location

unexplained.

molecular

cellular

the

fraction

levels

sub-cellular

fractionation

hepatoma

enzymes[ribonucleotide

deoxyribonucleotide

thymidine

as well

membrane

kinase,

as for

thymidylate

the high

molecular

DNA.polymerase We now report

proliferating rat

of cellular

a membrane

the predominant

(9),

from

of high

has been verified

The ambiguous

distinct

of Novikoff

has been further is

synthesizing

which

have shown that

microsomal high

is,

the presence

supernatant

(2-6).

be a consequence

significance (7,8)

cells

enzyme - that

- may not only

(1),

the post-microsomal

of eukaryotic

of this

siological

in

and Potter

spleen)

tissues contains

that

this

cytoplasmic

(Novikoff

hepatoma,

DNA which

can function

* This research was supported CA-11978 from the National

Copyright 0 1973 by Academic Press. Inc. All rights of’ reproduction irl my fcmn rcserwd.

membrane regenerating

as template

by Public Health Cancer Institute.

633

fraction

Service

from

rat

liver,

and/or Reseach

rapidly and normal initiator Grant

in no.

BIOCHEMICAL

Vol.54,No.2,1973

the

synthesis

rived

from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of polydeoxyribonucleotides. the

is virtually

corresponding incapable

In contrast,

normal

rat

of stimulating

liver

The cytoplasmic cribed

(8).

membrane

After

microsomal

overnight

supernatant, in the

is placed

a discontinuous

cribed

atop

by Murray

cytoplasmic Nucleic luted

1:lO

(1,000 with

--et al

membrane acid in

is

disc

are

acid-insoluble

method

of Bollum

de-

fraction

resuspended

volumes

Nucleic

acid

by Dounce

This (0.8

suspension

- 2.0 M) as des-

of this

fractions After

of chilled

the

are

di-

centrifugation

(-20")

sedimented

gradient,

1.3 M sucrose.

as follows:

10 min at 90".

des-

x g) of the post-

at approximately

four

ethanol

are mixed

by centrifugation

(15,000

in 10 mM NaCl.

is measured

into

is

gradient

prepared

resuspended

DNA synthesis Mann)

bands

extracts

membrane

as previously

(75,000

Upon centrifugation

fraction

the supernatant.

prepared

the supernatant.

sucrose

(11).

20 min),

30 min)

is

pellet

of

10% NaCl and heated

x g for

x g for

fraction

l/5

acid

DNA synthesis.

centrifugation

lower

nucleic

and Methods

the resulting

homogenization

cytoplasmic

--in vitro

Materials

the

by the incorporation

product

of dTMP-H3

by a modification

of the

(Schwartz-

filter

paper

(12). Results

Several RNA in the serve

reports

biosynthesis

to initiate

fraction,

previously

from

petence ponderance

this

involvement

for

and/or

its

a low but

ability

to stimulate

membrane initiator

nucleic

acid.

fraction for

634

nucleic

to test

amount

DNA synthesis.

DNA synthesis

this

(Table

of

Nucleic

demonstrated

acid

may

membrane

constant

has in fact

membrane In order

rather

of

which

The cytoplasmic

to contain

of RNA in the cytoplasmic

RNA as the active

the possible

DNA synthesis.

shown

cytoplasmic

as template

have indicated

of DNA by means of oligoribonucleotides

long-chain

RNA, has been tested acid

(13-16)

1).

comThe pre-

has suggested

hypothesis,

the

BIOCHEMICAL

Vol. 54, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I NUCLEASE PRE-TREATMENT EFFECT UPON ABILITY

OF CYTOPLASMIC MEMBRANE NUCLEIC ACIDS:

TO STIMULATE

DNA SYNTHESIS

CYTOPLASMIC MEMBRANE-ASSOCIATED NUCLEIC ACID

NUCLEASE TREATMENT

dTMP INCORPORATION (CMP)

No Addition

495

Novikoff

Hepatama

12,350

Novikoff

Hepatoma

RNase

Novikoff

Hepatoma

DNase

10,775 551

Normal

Rat Spleen

1250

Normal

Rat Spleen

RNase

1670

Normal

Rat Spleen

DNase

530

12-Hour

Regenerating

Liver

12-Hour

Regenerating

Liver

RNase

1010

12-Hour

Regenerating

Liver

DNase

580

1740

All assays are carried out in a two-step incubation. 0.1-0.3 A2~0 units of nucleic acid extract are incubated with and without nuclease or 30 min at 37" in 180 ~1 0.02 M glycine buffer pH 7.0. RNase-treated tubes contain 10 pg pancreatic RNase A(pre-heated to destroy DNase) while DNase: treated tubes contain 5 pg DNase and 0.8 pmoles MgC12. After the initial incubation all tubes are heated 5 min at 90" and, after cooling, 65 ~1 of DNA polymerase assay components are added: 1.25 pmoles 3.125 pmoles glycine buffer pH 8.0, 75 mumoles dithiothreitol, 78 MgCl mumo? es ' each of dATP, dGTP, and dCTP, 5 ~1 TTP-H3(0.5 mCi/ml, 17.3 Ci/mmole) and 100 pg Novikoff cytoplasmic membrane protein. After incubation at 37" for 60 min, acid-insoluble counts are assayed as described in Methods.

nucleic nucleases poration

acid prior

extracts

have been pre-incubated

to use in a DNA polymerase

of deoxynucleotide

by the action

of pancreatic

into

acid-insoluble

with assay

and without (Table

1).

The incor-

product

is

not affected

nucleic

acid

RNase A upon the membrane

635

specific

e';-

Vol. 54, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II COMPARISON OF EFFICIENCIENCIES NUCLEIC ACIDS INSTIMULATION

OF VARIOUS CYTOPLASMIC MEMBRANE-ASSOCIATED OF DNA SYNTHESIS

SOURCE OF MEMBRANE-ASSOCIATED

dTMP INCORPORATION CMP/A260 Units

NUCLEIC ACID

Normal

Rat Liver

310

Normal

Rat Liver

350

Normal

Rat Liver

240

12-Hour

Regenerating

Liver

1880

12-Hour

Regenerating

Liver

1920

12-Hour

Regenerating

Liver

2340

24-Hour

Regenerating

Liver

1330

36-Hour

Regenerating

Liver.

210

36-Hour

Regenerating

Liver

450

Rat Spleen

6700

Novikoff

Hepatoma

5300

Novikoff

Hepatoma

11,500

Novikoff

Hepatoma

16,500

Normal

All assays are carried out in 250 ~1 of solution containing the following: 1.25 moles MgC12, 3.125 moles glycine buffer pH 8.0, 75 mumoles dithiothreitol, 78 mumoles each of dATP, dGTP and dCTP, 5 ~1 TTP-H3 (0.5 mCi/ml, 17.3 Ci/mmole), 0.1-0.3 A 60 units of nucleic acid extract and loo-150 pg Novikoff cytop ? asmic membrane protein. Incubation is 60 min at 37". tracts

while

lation

of DNA synthesis

is

abolished

sensitive indicate which

DNase pre-treatment by Novikoff

by pre-treatment to RNase Tl,and

that stimulates

the nucleic

abolishes

cytoplasmic

with is acid

DNA synthesis

not

snake inhibited

from is

incorporation.

636

stimu-

nucleic

acid

venom phosphodiesterase, by RNase T2.

the cytoplasmic

DNA.

membrane

Also,

These

membrane

is data fraction

in-

BIOCHEMICAL

Vol. 54, No. 2,1973

We have also

observed

from

various

sources

plate

and/or

initiator

lated

with

liver

cytoplasmic

the rate

inactive

acid

correlation

liver

the

rate

of

maximal

phate.

acid

cytoplasmic require

activity

data

suggest

II).

Rat

spleen

activit,y.

membrane

has been found activity

nucleic

with

of the 24-

to the generally

template

corre-

to be consistently

and/or

12 hour and 36-

observed

initiator

"activated"

membrane

nucleic

all

deoxyribonucleoside

four

marginal

acid

compe-

calf

triphosphates with

incorporation

as Actinomycin

correspondence

one triphoswhile

the sulfhydrl

D are quite

between

thymus

has been made (Table

activity

affect

as well

a close

is

(Table

rat

utilizing

and have only

N-ethylmaleimide

ability

proliferation.

ATP does not significantly

inhibitor These

tissue

tissue

activity

of DNA synthesis

Both reactions

with

is contrary

nucleic

this

as tem-

have considerable

The declining

extracts

of membrane

DNA and Novikoff

for

extracts

has been observed

extracts.

A comparison

III).

Novikoff

acids

to serve

have been found

and low but consistent

regenerating

with

of the

extracts

nucleic

ability

and that

of proliferation

liver

membrane

in their

in DNA synthesis

activity

regenerating

tence

cytoplasmic

greatly

comparable

extracts

hour

differ

membrane

while

Intermediate

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibitory.

the two templates.

Discussion We have demonstrated Novikoff which

hepatoma, can serve

cytoplasmic

rat

membrane

proliferation

enzymes gested

spleen

as template

(17,18).

a complex

zymes may exist

and/or

initiator

also

contains

enzymes which

in the cytoplasmic that

the cytoplasmic

in this

membrane

and regenerating

fraction

and DNA synthesizing ular

that

all

Together membrane

fraction

of deoxyribonucleotide

637

for

liver

from

contains

DNA

DNA synthesis.

several

show definite with

fraction.

rat

fraction

deoxyribonucleotide responses

the predominance (9,10),

The

this

to cellof these

data

and DNA synthesizing

has sugen-

BIOCHEMICAL

Vol.54,No.2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE III COMPARISON OF ACTIVATED

CALF THYMUS DNA AND CYTOPLASMIC MEMBRANE NUCLEIC

ACID IN DNA SYNTHESIS

% CONTROL NOVIKOFF CYTOPLASMIC MEMBRANE NUCLEIC ACID

TREATMENT Complete

ACTIVATED

CALF THYMUS DNA

100

100

-dNTP

33

25

-2dNTP

23

15

-3dNTP

5

5

110

89

8

3

28

7

+ATP (2.0

mM)

tN-Ethylmaleimide +Actinomycin

All

(0.3

mM)

D (40 mg/ml)

assays

100% (activated

calf

100% (Novikoff

the exception

(19),

for

ponents,

of the

it

plasma

these

DNAs include

the associated

membrane

membranes

DNAs.

inherent

potential would

nucleic

for

be unwise

cytoplasmic

membrane

localization

in the

(20),

Hypotheses

= 2490 cpm

in cellular

artifactural to suggest

cell.

in association

and mitochondria

regarding

and information

that

has

transfer

procedures

the observed

of

(19). and

of cellular

com-

localization

of

reflects

the

What does seem to be of significance

638

With

the function

fractionation

enzymes

(21).

function

redistribution

DNA and DNA synthesizing intact

acid)

DNA, no physiological

gene amplification

problems

cpm

DNAs have been described

of the mitochondrial

been determined

Because

DNA) = 22,000

"cytoplasmic"

microsomes

these

thymus

cytoplasmic

Previously, with

out as in TABLE II.

are carried

"true" is

BIOCHEMICAL

Vol. 54, No. 2,1973

the possible enzymes well tween

association

uniquely

responsive

as the striking

and slowly

Further

investigations

DNA and a group

will

of DNA synthesizing

of cellular

in cytoplasmic

proliferating

proliferation,

membrane

DNA content

as be-

tissues. be required

to clarify

DNA and its

membrane-associated

to the deoxyribonucleotide

membrane

this

to the demands

difference

rapidly

the post-microsomal ship

between

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and DNA synthesizing

the

potential enzymes

nature

of

relationof this

fraction. References

1. Bollum, F.J. and Potter, V.R., J. Biol. Chem., 233, 478 (1959). G., Nature, Keir, H., Smellie, R., and Siebert, 196, 752 (1962). 32: Littlefield, J., McGovern, A., and Margeson, K., Proc. Natl. Acad. of Sci., '49, 102 (1963). 4. Kier, H. %'i "Proc.Nuc.Acid Res. and Mol. Biology", 4, 81 (1965). Biophys. Res. Comm., 39, 100 (1970). Friedman, D., Biochem. 2: Weissbach, A., Schlabach, A.,,Fridlendor, B.,and Bolden, A. Nature (New Biol), 231, 167 (1971). 7. Baril, E., Jenkins, M., Brown, I?., and Laszlo, J., Science 169, 87 (1970). 8. Gil, E., Brown, O., Jenkins, M. and Laszlo, S., Biochem., lo. 1981 (1971). 9. Baril, E., Baril, B., and Elford, H., Proc. Amer. Ass. Cancer Res. l3-, 84 (1972). 10. Elford, H., Luftig, R. and Baril, E., "Abstracts Ninth Int. Cong. Biochem." (Stockholm) in press. 11. Murray, R., Suss, R., and Pitot, H. in "Methods in Cancer Research", H. Busch, Ed., 2, 239, Academic Press, N.Y. 12. Chang, L.M.S. and Bollum, F.J., J. Biol. Chem., 246, 5835 (1971). 13. Verma, I., Meuth, N., Bromfeld, E., Manly, K.and Baltimore, D. Nature (New Biol.), 233, 131 (1971). 14. Brutlag, D., Schekman,R. and Kornberg, A., Proc. Natl. Acad. Sci. 3, 2826 (1971). 15. Sugino, A., Hirose, S.sand Okazaki, R., Proc. Natl.Acad.Sci. 69, 1863 (1972) 16. Chang, L.M.S., and Bollum, F.J., Biochem. Biophys. Res. Comm. 4G, 1354 (1972). 17. Baril, E., Baril, B., Elford, H. and Luftig, R. in "DNA Replication and the Cell Membrane", El. Kohiyama and A. Kolber, Eds. (1973) in press. 18. Baril, E., Baril, B., Luftig, R. and Elford, H. Submitted for publication. 19. Bell, E., Nature, 224, 326 (1969). 20. Lerner, R., Meinke, W.and Goldstein, D., Proc. Natl. Acad. Sci. 68, 1212 (1971). 21. Kalf, G., Biochem., 3, 1702 (1964).

639