Journal of Histochemistry & Cytochemistry

Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques. H G Gratzner, A Pollack, D J Ingram and R C Leif J Histochem Cytochem 1976 24: 34 DOI: 10.1177/24.1.815428 The online version of this article can be found at: http://jhc.sagepub.com/content/24/1/34

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THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1976 by The

AND

. Inc.

Societ

DEOXYRIBONUCLEIC

ACID

CHROMOSOMES

or

Cancer

curred.

The

antibodies

jugate

between

in

to BrdU

bovine

which

were

serum

Institute,

(BrdU)

of chromosomes

SINGLE

produced

albumin

and

1, pp. 34-39, 1976 Printed in U.S.A.

CELLS

R

AND

Miami,

AND

novo

C. LEIF

Florida

33123

or iododeoxyuridine

de

24, No.

TECHNIQUES’

D. J. INGRAM

Research

to 5-bromodeoxyuridine

regions

IN

IMMUNOLOGIC

A. POLLACK,

Papanicolaou

Antibodies

REPLICATION

BY

H. G. GRATZNER,

cells

Vol.

CYTOCHEMISTRY

Histochemical

may

deoxyribonucleic

in rabbits

by injection

bromouridine

be used

acid

to identify

synthesis

has

of the antigen,

(BrU),

or

oc-

a con-

iodouridine.

Specific

antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase

The tion

methods.

Chromosomes

cells

not

containing

accomplished

in

techniques

(11)

graphic

The periods

of

poses

to

detection.

of

graphic

and

exposure

for

techniques

The

replication

method

specific

are

for

in

the

the

The as

and

potentially

ysis

of

DNA

as

for

of

an

groups the

the

was

was

supported

NIH5501RR0569003, Grant CA13441. 2 Abbreviations: phosphate-buffered 5-bromo-2-deoxyuridine;

BSA,

bovine

dine coupled BSA; BSA-T, immunoglobulin

NO1CB43962 Research National

by

DNA, deoxyribonucleic acid; saline; TdR, thymidine; IdU, 5-iodo-2-deoxyuridine;

serum

albumin;

to BSA; thymine G.

BSA-IU, riboside

BSA-BrU, iodouridine coupled

mixture

at pH

9.5 and

at 9.5

to a stable stand

for

and

amino

stirred

3-OH

groups

was then

added

is unstable,

150

for reduction

added and

mg

the

36 hr against

cold

the

sodium

of

mixture

Five ml formic was adjusted to pH The

the

Since

were

later.

of

to 10

for 45 mm;

5% K2CO3.

with

1 hr

riboat room

acid

18 hr.

hydroxide for

2’-

C

(4):

thymine

the

conjugate,

the solution

dialyzed

or

ex-

Beiser

periodate

to the

conjugate

to

and

oxidize

This

con-

prepared

8.5

was

were with

preparation running

1 was

water

and

lyophilized.

mg/ml

equal

Health

BSA-BrU

volume

(Difco),

Cancer

of

BSA

complex

then

and NO1CB33861 Support Grant Institutes

to

the

allowed

ultrastructural

National

BSA.

mg

Erlanger

coupling

maintained

Four work

order for

in 10 ml H2O

an ‘This

in of

were

M sodium

in 0.1

of ribose

added and M sodium

anal-

of

ribonucleosides

iodouridine

oxidized

BSA-nucleoside

to

The albumin

procedure

banding.

METHODS

serum

borohydride

level.

Institute Contracts and by General

exhibit AND

bromouridine,

lysines

pH

microscopy for

the

ml of 280

have

detect-

5-bromo-

field

not

methods: bovine

temperature

antibody

microscopy at

were

is applicable

bright

electron

mg

chromosomes.

analogue,

synthesis

100 side

pur-

We

and

technique

well to

applicable.

use

thymidine

deoxyuridine. fluorescence

cells

the

by

lack

autoradio-

technique

to

long silver

for

analysis.

not

involves

sufficient

addition,

an immunologic

DNA

however,

produce

flow

jugated actly

relatively

did

Immunologic

autoradio-

3H-thymidine.

require

In

automated

developed

by

methods,

resolution

grains

past

utilizing

autoradiographic

cytologic

the

BrdU

MATERIALS

identification of those cells in a populawhich is undergoing DNA2 replication has

been

ing

from

and

each

rabbit

were

bled

1 ml

4 days sera

diffusion by

(Sigma) dure

injected

for 8 weeks.

to the for

final

into

Animals

injection.

antibody

(8) and

chromatography:

chromatography

macia)

coupled to to BSA: IgG,

fixation

was

with

Adjuvant

The

activity

Ouchterlony

by double

(16).

Affinity

bromouri-

emulsion

tested

emulsified

Freund’s

week

subsequent

microcomplement

PBS, BrdU,

the

other

were

were

Complete

of

every

undiluted

in saline

of

to which had described

Antibody on

5-bromouridine

been by

34


covalently Eichler

was

AH-Sepharose

4B

or thymine coupled

and

Glitz

by (3).

The

purified (Phar-

riboside the

proceglobulin

DNA

fraction

of antiserum,

REPLICATION

obtained

by adding

BY

IMMUNOLOGIC

(NHJ2SO4

to serum to final concentration of 40% at 4#{176}C, was applied to the column in a volume of 5-10 ml 0.05 M Tris-HCI buffer, pH 7.2, 0.15 M NaCl. The column was

then

washed

with

the

same

buffer.

The

column

was then eluted with 0.1 M acetic acid, 0.15 M NaCl or with bromodeoxyuridine (50 g/ml). Relevant fractions were precipitated at 40 (NH4)2SO4 (4#{176}C). The

35

TECHNIQUES

saline.

The

cells

(3:1),

placed

on

were

fixed

slides

in

and

methanol:acetic

air

dried.

acid

The

slides

were

heated

at 65#{176}C for 1 hr in 95% formamide, 5% saline acid, 0. 15 M sodium chloride) in order to denature the nuclear DNA (2). The slides were then coated with normal goat serum for 15 mm, rinsed in PBS and coated with anti-BrdU antibody.

citrate

(0. 15 M citric

After

incubation

at

room

temperature

for

30

mm,

Cell culture: The continuous cell line, CHO, obtained from Dr. D. F. Peterson, Los Alamos Laboratories, was used for these studies. The cells were maintained as monolayers in Ham’s F-b medium (Gibco), supplemented with antibiotics, 10% calf

slides were rinsed in PBS twice for 5 mm/rinse and coated with fluorescein-labeled goat anti-rabbit IgG (Hyland Laboratories; diluted 1:40). In some experiments, the fluorescein-labeled goat IgG was replaced with goat anti-rabbit IgG labeled with horseradish peroxidase (Miles/Yeda) diluted to 1:500. The slides were stained for 5 mm with a solution of 75 mg 3’, 3”-diaminobenzidine (Sigma) and 0.003% H2O2 in 100 ml PBS (10) for histochemical localization of the peroxidase. Chromosomes from CHO cells were prepared by incubating cell monolayers in 0.1 zg/ml

serum

colcemid

precipitate

was

against

Tris

redissolved

or

nuclei

overnight

Autoradiographic

by dipping into

exposing

dialyzed

buffer.

Autoradiography:

performed

and

the

and

Kodak slide

5%

analysis

the microscope NTB

for

7-10

fetal

slides

nuclear

was

with

cells

emulsion

and

days.

calf

serum.

Incubation

was

in a

CO2 atmosphere. Cell synchrony was induced by the isoleucine starvation method of Tobey (20), but the cells were grown and synchronized as

95%

air,

5%

monolayers

in

100-mm

Petri

plates.

Pulse

was accomplished by medium used to reverse

removing the the isoleucine

washing

Hanks’

and

the

adding

cells

with

10 ml

supplemented

with

Eagle’s 10MM

fluorodeoxyuridine. for

1-hr

washed

and

after

suspended

plates which

salt

essential

3H-BrdU

The

intervals

balanced

minimal

(1 MC/ml) were

the

in cold

labeling

complete starvation,

then

cells

were

plates,

for

2 hr.

Cells

were

swollen

in

0.075

M

acetic acid (3:1), subjected to the above.

scraped

spread

on

antibody

fixed

from in

slides,

air

procedure

the

methanoldried

as

and

described

F-b RESULTS

and solution

Unfractionated lenged with

medium + 1

MM

incubated scraped,

phosphate-buffered

(Fig. well

lipes

by

from rabbits chalBSA-IU produced

or

double-diffusion

1). Bands are produced as BSA and BSA-BrU.

ment

fixation serum

studies,

for

the

titers

determined

various

incorporated

into

the

as titer

antigens.

yielding

fixation, the titers with BSA alone, 1/50; denatured BrdU

analysis

against BSA-T By microcomple-

we

against

example,

BSA

antisera BSA-BrU

precipitin

of

BSA-BrU

then KC1,

70%

For

complement

BSA-BrU DNA,

were 1/400; 1/50 and for

denatured

DNA,

1/2400

(6). Cross-reaction could be eliminated of

this

antiserum

mouridine elution

of unfractionated globulin from with BSA-BrU. Ten M1 of antigens placed in the peripheral wells; 10 Ml

undiluted globulin fraction (AB) were placed in the center well. Plates were incubated 24 hr at room temperature, dried and stained with Coomassie Blue stain.

with

double

diffusion.

results

the

column antibody

of elution

thymidine

column.

bromodeoxyuridine was

to

produced


fractionation analyzed

by

eluant

BSA-IU

by affinOuchter-

globulin

fraction

BSA

from

(50 mg/mI). with

which

BSA-BrU,

of antibody

bro-

acid-NaCl

(6).

specifically (6). Figure The

which

a preparation

The

antibodies

to Acetic

BSA-T

The results of antibody chromatography were

BSA-T; anti-BrdU

tity

Sepharose coupled.

yielded

no band

contained 1. Analysis rabbits immunized (0.5 mg/ml) were

been

of antibody

lony

FIG.

BSA and with thymidine by affinity chromatography

on

had

displayed ity

with

and

removes

the

2

the

shows

a Sepharose-

in

this

A band

case

was

of iden-

in addition

to

36

GRATZNER

ET

AL.

ide

vapor.

enables and

those

Figure

4 shows synchronized

during

#{149}

which

which labeled

.,

Counterstaining

with

a differentiation

for the

1

have the

not

hr

with

green

positive

incorporated

results CHO

course

methyl

between of an cells BrdU

of S phase.

cells BrdU.

experiment have

at

in

been

various

Comparison

pulse times of the

It

4.

- ( 2.

FIG.

from

Analysis

of

fractionated

Sepharose-thymine

g/ml bromodeoxyuridine. in Figure 1.

precipitin (17)

lines

also

reacts

with

with

BrdU,

beled

with and

with

dicative 3). When

of BrdU BrdU

et

found

cross-reactions cells

subsequently

goat

the

Sawicki antibody

When

by

fluorescein,

50

as described

al.

crossof

BSA-T.

are

purified

with

anti-IdU

studies:

BrdU

antibody dures

and

with

Cytologic

treated

BSA-BrU.

that

eluted

column

Plates

reported

anti-BSA-BrU

antibody

riboside

one

the

into was

x 250.

with

above

proce-

IgG tagged fluorescent,

become

incorporation at 50 g/ml

la-

treated

of

anti-rabbit

nuclei

pulse

FIG. 3. Demonstration of anti-BrdU fluorescence in nuclei of pulse-labeled CHO cells. Cells were synchronized and treated as described in Materials and Methods. Pulse of BrdU was for 1 hr, 6 hr post-isoleucine reversal. Photography was on High Speed Ektachrome film. Arrow indicates an “unlabeled” cell.

with in-

DNA

(Fig.

added

to

Immuno.

the -

antibody fluorescence els, of

preparation prior to staining, was decreased to background

whereas

the

thymidine

addition

had

of this

no

effect.

the lev-

not

Scm). .

U

concentration

Cells

2Autorad.

C

pulsed C’)

with

BrdU

were

background

not

fluorescent,

fluorescence,

as

or had shown

by

very

low

the

nu-

cleus indicated In a previous

by the arrow in Figure 3. publication (6), we showed

labeling

of nuclei

indices

this immunofluorescent comparable to 3H-TdR

immunologic to bright-field

tution

of

the

cells

by

technique microscopy

goat

conjugate with with horseradish stained

E a.

that

be performed with

S

by

results utilizing

or 3H-BrdU.

The tended

those

could

technique, autoradiography

0

anti-rabbit

IgG-fluorescein

goat anti-rabbit peroxidase. With

which

gray-brown.

treatment

has been exby the substi-

of the

have

The slides

IgG this

incorporated

contrast with

labeled reagent, BrdU

was osmium

Hours FIG.

are

enhanced tetrox-

4.

Comparison

of

labeling

index

determined

by immunoperoxidase-anti-BrdU (Immuno) and autoradiography (autorad). At least 500 nuclei were counted for each time point. Cells were synchronized as described in Materials and Methods. 3H-BrdU incorporation was also monitored by scintillation counting (Scint.) and plotted as cpm/106 cells.


DNA

REPLICATION

BY

IMMUNOLOGIC

or

37

TECHNIQUES

pulsed

cells

anti-BrdU

incubated

antibody

with

are

not

PBS

lustrated in Figure 5. The cells 1 hr, 9 hr post-isoleucine reversal, about labeling

c #{149}

S.

some

banding

nique

(Fig.

by Sc).

the

We

patterns

technique

with

is il-

were pulsed for or at a period 3H-TdR chromo-

immunofluorescent have

not

obtained those

or quinacrine

of

This

into S phase, as shown by (Fig. 4). We have also obtained

1

banding

a

instead

banded.

yet

by

techcompared

the

produced

the

immunologic by

the

Giemsa

of

monitoring

methods.

DISCUSSION

The

immunologic

DNA

synthesis

IdU appear applicable volving

methods with

antibody

to have to several

areas

replication,

tion S

of

cells

phase

both

varied of tumor

well

as

tems

the

of

cence

C 5. Effect of anti-BrdU antibody treatment on metaphase chromosomes pulsed with BrdU in S phase (9 hr post-isoleucine reversal). Immunoperoxidase technique: a, control chromosomes, no BrdU; b. 10 MM BrdU, 1-hr pulse; c, similar treatment, but by immunofluorescence. FIG.

results with that

of the anti-BrdU peroxidase conventional autoradiography the

immunologic

labeling

kinetics

technique in a manner

diographic methods Chromosomes, chromosomes labeled treated

from with by

chromosomes

cence and chromosomes

technique indicates can

similar

be

used

CHO

cells

which

When were

appear

banded,

immunoperoxidase from cells

not

both

by

exhibited mixed

ploidies,

pulse

binds

assigned content

measurement

quantitative

fluores-

specifically

to DNA

to S phase lies between

by G, and population

sysstages

are the

those DNA

G2. It is obvious that, of cells of different

measurements

may

be

misleadthe

in-

3H-TdR and autoradiography the detection of S phase cells, are not applicable to flow systems. The immunologic methodology

for

methods,

which

in this

develop S phase

a fluorescent

technique

which

be

the

ments,

encountered

report

was

could

spectroscopy.

we most

with


in

the these

in order

suspension

cytoplasmic methods.

to

for determining to

flow

preliminary

specifically

cells of

explored

applied In

have

BrdU-labeled nated

exploit

of

described

cence

methods. Control pulsed with BrdU

the

flow

specific

ing.

are

fluores-

a

con-

employ

upon

which

such

in

of cells

population

in

by

Conventional

for

BrdU for 1 hr during S phase the immunologic procedure,

levels in a

which

depend

cells DNA

corporation

to autora-

with 3H-TdR. preliminary results:

(1, 5). The cells whose

are with

be heteroploid as such as lympho-

of cells

content

of a dye

which

is dealing

The

cell

popula-

or fibroblasts.

cycle,

DNA

a

consisting

content.

determination

cell

for in

tumors one

techniques,

for

and

analysis

cells which may diploid cells,

normal

inand

research

cells

population

macrophages

of the

flow

from

DNA

Current

basic

those

when

cell

sists

cytes,

1

by

dispersed

with

or

of investigation in

of

is difficult

heterogeneous

techniques

of semiconservative

clinical analyses. Labeling indices kinetics: Detection

b

BrdU

as

potential

determination

repair

against

fluoresexperi-

stained

whole, and

elimi-

fluorescence However,

we

38

GRATZNER

have not writing. For

performed

cell

microfluorometry

kinetic

statically

studies

on cells

immunologic

which

fixed

also

has

techniques,

production

of silver

kinetics tumor

is used chemotherapy

results

of the

slides,

the

grains.

for

Thus,

than

the

number as

be

by

of

fluorometry

be to

DNA,

and

chromatin. number body

sites

molecule

molecules

and

the

DNA

replication

in

gaining replicons

utility

the

can

be

of

accessibility

of

BrdU,

which

associated

performed

effects

on

derived needle

the anti-

the

tissue

netics

vitro from

Chromosome of the

various

(11),

nism

the

technique

The

has

of

recently

been

identifying

focused

reflects individual

shown early

chromosomes

or

(7)

as

by

poly(A)

.poly(T) banding

sensitive

well

would

extend

should

be

as

sequences

Latt’s

possible

the A

regions

asymmetric

(9). positive

The should

his

authors

wish

to

photographic

this

chromoanalogues.

possibility,

exstaining

thank

Edward

Kramer

assistance.

rich

band

as resolu-

Exp

Cell

Res

74:288,

1964

Jotz MM: Deoxyribonucleic for automated cytology. 22:470,

acid cytoJ Histochem

1974

6. Gratzner HG, Leif RC, Ingram DJ, Castro A: The use of antibody specific for bromodeoxyuridine for the immunofluorescent determination of DNA replication Cell Res

it

RA:

chromosomes.

Cytochem

more

Tobey

3. Eichler DC, Glitz DG: Nucleotide-specific antibodies as potential blocking agents in the structural analysis of nucleic acids. Biochim Biophys Acta 335:303, 1974 4. Erlanger BF, Beiser SM: Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc Natl Acad Sci USA 52:68,

immunobe

HA,

metaphase 1972

5. Gill JE, chemistry

in

CITED

Cell cycle analysis in 20 minutes. Science 184:1298, 1974 2. Dev VG, Warburton D, Miller OJ, Miller DA, Erlanger BF, Beiser SM: Consistent pattern of binding of anti-adenosine antibodies to human

of

fluorescence,

inasmuch higher

and base

(19).

in

synthe-

possibly

results, to gain

mechabanding

to be valuable

which a

ki-

DNA replication chromosomes has

regions,

yields

or con-

introduction for karyotyp-

on

exploring

level

stains such it should be

peroxidase-antiperoxidase

1. Crissman

data

cell

replicating

technique, and

the Latt

late

in heterochromatic

logic

reflect

phenomena.

immunologic

microscopic

replication utilizing

LITERATURE

toxic that

the

immunocyto-

the

ACKNOWLEDGMENT

15).

Since

the

BrdU

of sections with BrdU

techniques

banding

which the

patterns

sis

studies:

attention

of

(14,

banding

of

anticipates

medium

in vivo

DNA

currently

BrdU

negligible

incubation tumors

culture

obtained

with

of the

electron

for

that as well

predicated

incorporation and

patient,

is

are

tool

identi-

(13), since heavy metal can be utilized. Thus,

procedure

in antibody

technique

in vivo

from in aspirates

taining

ing

We

a useful

extends

the

to study ultrastructure,

ploiting

with

adjacent

problems

feasible some

of visualizing

prove

use

be

obtains,

of chromosomal

The

of resolution as osmium

to

latter

the way, for example, into chromosomes,

method

to

whether

that

to a new

methodology

hope to determine of BrdU bound per

this

concept

insight into are packed

tech-

chromosome If the

also

which

anti-C antican be found

technique

may

The micro-

entire

banding

quinacrine

anti-A and conditions

immunologic

statistics. by

the regions

or

signal enzyme it will

to

antibody.

fication (12). Ultrastructure:

for

The

the

to the

then

binding.

upon

enable

and

whether

Giemsa

niques, as shown with body (2, 18), or whether

on

the

the

technique

by

steric

by

peroxidase

protein

also

present

band

as lead

pulses by indirect Obviously,

is restricted

high

incorporated

We also of molecules

also

which

since

binding

by

anti-BrdU

accessible

by accu-

BrdU

to determine

with

of the

degree of denaturation of the of other factors, such as possible

also of

be necessary

cell

of the

masking

shorter

can be gained methodology.

sufficiently

Poisson

affected

the

through

course (13), rapidly techniques.

grains, be

antibody

will

antibodies

is a function

of

should

unaffected

quantitation

the

counting

tion

when

synthesis as measured is potentially more

of photons

to

long for

treatment

cell proliferation would be available comparison to the autoradiographic

rate

anti-

relatively necessary

of a particular

of DNA

the over

the

monitoring the or radiotherapy

effects

Quantitation immunofluorescence

performed

in that

AL.

amplification histochemical

advantages

body method does not require exposure times and manipulations the

are

microscope

on

method

autoradiographic

as of this

ET

7. Latt SA: bonucleic

in single 95:88, 1975

cells

Microflurometric acid replication


and

chromosomes.

detection in human

Exp

of deoxyrimetaphase

DNA

chromosomes.

Proc

Natl

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Journal of Histochemistry & Cytochemistry

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