0022-1554/79/2712-1655$02.OO/O THE

JOURNAL

Copyright

OF

Laser

AND

HISTOCHEMISTRY

Histochemical

© 1979 by The

Flow

Cytometry

of Cytokinetics,

12,

pp.

Inc.

1655-1656,

Printedin

Cancer Anthracyclines

Chemotherapy: Detection by Flow Cytometry’

and

AWTAR

Division

Vol. 27, No.

CYTOCHEMISTRY

Society,

KRISHAN2

Comprehensive

Cancer

AND

Center Medical

for

RAM

the

GANAPATH!

State

ofFlorida

Miami,

School,

(BR

Florida

and

1979 U.S.A.

of Intracellular

79-187)

Department

ofOncology,

Univer8ity

of Miami

33101

The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracyclime group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference ofthese compounda with the binding ofpropidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence ofthese antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use ofthese two methods for intracellular detection and quantitation of a number of important anthracydlines.

Flow

cytometry

in cell (8).

Recent

cycle

(FCM)

biology,

studies

analysis

cancer

cell

have

the

to a number

kinetics

and

demonstrated

(3, 5), aneuploidy differential

the

detection

In our

chemotherapy.

monitoring

has contributed

immunology,

use

of FCM

we

of

used

and

cancer chemotherapy (6). As most of the cancer chemotherapeutic agents specific cytotoxic effects, it is essential to study not erative

binding

status

of the

target

tissues,

but

also

the

is

rapid

FCM

tumor

have only

Intracellular presence of anthracyclines, can be detected in FCM by both indirect and the direct method. The indirect method is based on the observation that fluorescence of ADR is quenched on binding to DNA. This reduction in fluorescence can be quantitated with either a spectrofluorometer or in a laser flow system. l.a contrast to ADR, fluorescence of propidium iodide, is enhanced approximately 20-fold on binding to double stranded nucleic acids. Exposure ofcells to ADR before staining with P1, decreases the amount of nuclear fluorescence. This reduction in fluroescence is influenced by intracellular transport, retention and effiux of ADR as well as by the extracellular drug concentration and the length of exposure (7). With this indirect method, we have detected intracellular presence of a number of anthracyclines and their metabolites both in vitro and in vivo. As shown in Table I most of the anthracyclines studied had profound effect on the fluorescence of cells stained with propidium

cell

in scheduling

have

normal

RESULTS

chemotherapy

(1, 2), and

laboratory,

response

of advances

cancer

of for

cells

to

cell cycle the proli.f-

intracellular

transport,

and effiux

of the chemotherapeutic agent. Some of our recent work has focused in this area (7), and the present report demonstrates the potential of FCM for detection and monitoring of intracellular anthracyclines (e.g. adriamycin and its related anthracydlines) in individual tumor cells. MATERIALS

AND

MITh’HODS

iodide.

Human leukemic lymphoblasts ofCCRF-CEM cell line were grown in Eagles S-MEM medium supplemented with 10% fetal calf serum and antibiotics penicillin and streptomycis. P388 leukemic cells were obtained from DBA/2 mice. Samples

of

adriamycin

hydrochloride

(ADR,

NSC

123127),

N-

trifluoracetyladriamycin-14-valerate (AD-32, NSC 246131) and N-trifluorocetyladriamycin (AD-41) were generously provided by Dr. Mervyn Israel. Rubidazone (NSC-164011), nogalamycis (NSC-180024), daunomycis (NSC-82151) and carminomycin (NSC-180024) were obtamed from the Investigational Drug Branch, DCT, National Cancer Institute. Stock solution (1 mg/mi) were made in 10% Tween 80 and further diluted with normal saline. Propidium iodide staining solution was made in 0.1% sodium citrate as described earlier (5). This solution is henceforth referred to as P1/citrate staining solution. For flow cytometry (FCM) samples were analyzed on a Coulter Electronic TPS-I cell sorter (Coulter Electronics, Inc., Hialeah, Fla.), equipped with a Spectra-physics Model 160-8 Argon ion laser. Fluorescent signals of cells analyzed at the approximate flow rate of 1O cells/sec. were stored a pulse height analyzer and recorded on a xy plotter.

Supported by the National of Health, Grant CA 23688. 2 To whom reprint requests University 33101.

ofMiami

Medical

Cancer should School,

Institute,

National

Institutes

be sent

at: R-71,

Cancer

Center,

P. 0. Box

016960,

Miami,

Florida

Data in Figure 1, show that P1 fluorescence of P388 murine leukemic cells exposed to various concentration of ADR for 3 hr before staining with P1, is related to the drug concentration. Thus in cells incubated with 50-100 g of ADR, there was approximately a 5-fold reduction (peak channel 7 to 15) in the amount of fluorescence of P1/ citrate stained cells. This quenching ofPl fluorescence can be detected both in vitro and in vivo. In mice injected with 4 mg/kg of ADR, spleen and tumor cells (P388) show maximum quenching of Pl-fluorescence. Besides

this

indirect

P1

interference

method

for

anthracycline

detection, we have recently used a Coulter Electronics TPS-I cell sorter and a Spectra.Physics 2-watt argon ion laser to directly excite and quantitate the intracellular fluorescence of various anthracyclines. An example of this is shown in Figure 2 which 8hows the drug fluorescence profiles of P388 tumor cells incubated with ADR, its analog AD-32, and AD-41, an intracellular metabolite of AD-32. Individual examination of cells, incubated with 10-100 pg/mi of ADR under a UV-microscope, indicated that there was a gradual appearance of intracellular fluorescence. This was confirmed by the FCM analysis. In contrast to ADR, AD-32 fluorescence could be detected both under the IJV microscope and in FCM, within minutes of drug (1-10 pg/mi) exposure. Cells incubated with AD-32 were highly fluorescent with peak channel value of 120 and absence of nonfluorescent cells in the sample. In contrast, cells incubated with ADRand AD-41 were less fluorescent (peak channel 50) and had a minor peak of non- or less fluorescent cells recorded in channel 10 (Fig. 2).

1655

Downloaded from jhc.sagepub.com at University of Reading on January 3, 2015

1656

KRISHAN TABLE

Interference

ofAnthracyclines

AND

LASEREXCITATION OFANTORACYCLINES

I

with

Propidium

CCRF-CEM

Iodide

Staining

of

Cells lOgLg/rnl

1,Lg/ml

1OOLg/m1

Adriamycin

91”

94

39

Rubidazone

88

76

39

Nogalamycin

88

76

48

Daunorubicin

85

76

39

Carminomycin AD-32

79

70

‘I

Gi

shifts

channel

27 112

106

100

peak

GANAPATHI

as percentage

of control.

e

21

41

H

II

100

120

RELATIVEINTIACELLILARFLOI1ESCENCE

2

EFFECT IF AIR IN FLUIRESOF PI#{149}STAINEI NUCLEI a zz z : -

-.

FIG. 2. Fluorescent profiles of P388 cells incubated in vitro for 4 hr at 37#{176}Cin ADR, AD-32 and AD-41 and directly excited with the laser beam. Cells incubated with ADR and AD-41 had a bimodal distribution with a minor peak at channel 10 and a major peak between channels 40 and 50.

I

1NI III

4-,

III

_

411

to study

the

Similar

studies

intracellular

daunorubicin,

1

FLISIESCEICE

from

be a fruitful correlate

FIG. 1. The fluorescent profiles of CCRF-CEM lymphoblasts incubated with various concentrations of ADR for 3 hr before their staining with P1/citrate. Note the ADR concentration dependent shift in the modal peak channel value.

on

area

in the

of important by

the

cells

study

on

these

Our

report

the

agents

rapid

also

for

cell the

With

cycle

analysis

cytochemical

basis

it is possible

or other

biochemical

on the interference the

FCM

not

probes

or

to detect

alterations.

of DNA

quantitation

only

binding

of cellular

chemotherDNA

content

can

been

recently

also

recently

be

used

shown

with

other

by Preisler

fluorochromes,

As

reported

in the

e.g.

Hoechst

33342,

(9).

The direct excitation of intracellular exploiting their fluorescent characteristics analogs.

nogalamycin.

e.g., A similar

present

study,

drugs is possible by either or by using fluorescent we have used this method

as

of these

preliminary

studies

that

this

can

further investigation as it may allow us to drug content of individual cells with the effects

CITED

of human solid 38:3333, 1978

P, Fredenkson

P, Kjaer

microfluorometry

classification

extended to in vivo samples where similar quenching of fluorescence was detected in various normal tissues (e.g. spleen, liver and tumor cells) after a single injection of 4 mg/kg of ADR. Our current work is focused on isolating from these drug treated populations subsets based on their individual drug contents. This technique have

and

B, G#{246}hde W, Johnston DA, Smallwood L, Schumann J, B, Freireich E: Determination of ploidy and prolifera-

characteristics Cancer Res

Flow

and quantitation

detection

proper

(3, 5), but

and of intracellular drug content but also to sort of their individual drug content and further drug,

clonogenicity with

rubidazone

anthracyclines,

proliferation.

2. Bichel

only

agents.

amount

for

not

of the

observations

apeutic

for

present

excitation

quantitate out

tool

anti-cancer

direct

of anthracyclines.

to other

LITERATURE

tive etry.

DISCUSSION

most

for

intracellular

their

1. Barlogie Drewinko

as shown

of a number

extended

carminomycin,

It is evident

1EL*IIVE*11111 II

is an ideal

been

approach has recently been used by Kaufman et al. (4) who used flow cytometry to study the intracellular distribution of a fluorescent methotrexate derivative.

ill

FCM

distribution

have

of human

and

tumors

T, Thommesen

transrectal

prostatic

by pulse

P, Vindel#{248}vLL:

fme-needle

carcinoma.

cytophotom-

Cancer

biopsy in the 40:1206, 1977 in 20 minutes.

3. Crissman HA, Tobey RA: Cell cycle analysis Science 18:1297, 1974 4. Kaufman BA, Bertino JR, Schimke RT: Quantitation folate reductase in individual parental and methotrexate

murine

cells.

J Biol Chem

253:5852,

of dihydroresistant

1978

5. Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188, 1975 6. Krishan A, Pitman SW, Tattersall MHN, Paika KD, Smith DC, Frei E: Flow microfluorometric patterns of human bone marrow and tumor cells in response to cancer chemotherapy. Cancer Res 36:3813, 1976

7. Krishan analogs

A, Ganapathi RN, Israel M: Effect of adriamycin and on the nuclear fluorescence of propidium iodide-stained cells. Cancer Res 38:3656, 1978 8. Melamed MR. Mullaney PF, Mendelsohn ML: Flow cytometry 9.

and sorting. John Wiley Preisler HD: Alteration 33342 to human leukemic 62:1393, 1978

Downloaded from jhc.sagepub.com at University of Reading on January 3, 2015

and Sons, New York, N.Y., 1979 of binding of the supravital dye Hoechst cells by adriamycin. Cancer Treat Rep