Use of plant lectin-induced agglutination to detect alterations in surface architecture of sarcoma 180 caused by antineoplastic agents

Biodmblpburmcdqy.

Pa#unorlPm& 197s.

VoL24. pp.1149-1152.

Printed ia Great Britain

USE OF PLANTLECTIN-INDUCED AGGLUTINATION TO DETECTALTERATIONS IN SURFACE ABCHITECTURE OF SARCOMA 180 CAUSEDBY ANTINEOPLASTIC ACENTS

Kou M. Hwang and Alan C. Sartorelli Departxmnt of Pharmacology,

Yale University

New Haven, Connecticut (Received

Considerable

evidence

normal cells

and cells

and chemical

carcinogens

clinically

useful

exists

suggesting

(1.2).

Little

antineoplastic

by chemotherapeutic either

cancer cells.

the capacity

surface

utility

reagents

to probe the surface

of these agglutinins

coqonents

cell

of the cell

surface

the role

of surface

cellular

transport

of concanavalin tions

architecture

Tumr cells after

the intraperitoneal

to reeve

from the ascitic

injection

contaminating

erythrocytes.

ad&d to the cell

suspension

Sarcoma 180 cells both uutreated

were treated

and drug-treated

0.9% NaCl, suspended in this tination

using either

ology employed allowed

cells;

solution

the changes in constituents and transformation;

(WCA) to detect

reports

of

the use

drug-induced

altera-

cells. of female CD-l mice 7 or 8 days cells.

(pH 7.2)-0.9%

The cells

were washed

NaCl per ml of packed

were suspended in Fischer’s Antineoplastic

and sealed

periods

were washed with Ca”, at a concentration

vessels

were

of tine.

Following

Control

incubation,

Mg++-free phosphate buffered-

of 1 x lo7 cells/ml,

Con A or WGAwas performed as previously an assessment of the rate and extent

medium

agents were

reaction

in the absence of drugs.

1149

The

and swvemmt of the

shaking water bath for various

cells

cells.

This commmication

concentrations,

identically

which possess

and to cause agglutination,

of 3-S x lo6 cells/ml.

at various

at 37’ in a gyrorotatory

Cells

changes

phenomena; and the location

fluid

volumes of 5 IPMTris-HCl

lectins,

of eukaryotic

of 6 x lo6 neoplastic

+ 10% horse serum at a concentration

incubated

(3).

or indirectly,

function

recognition

has been swmmrized

were isolated

viruses

and (b) existing

differentiation

membrane of Sarcoma 180 ascites

2 to 3 times with lo-15 cells

in cell

cycle,

A (Con A) and wheat gem agglutinin

in the surface

sites

of normal and neoplastic

macromlecules sites,

The plant

receptor

of plasma membranes that occur during the cell

attack

directly

the structure,

to assess:

by oncogenic

is being expended to determine whether

agents create,

to bind to specific

in plasma me&manes between

to transformation

effort

can be exploited

20 February 1975)

differences

in the plasma membrane of susceptible

are important

06510

24 January 1975; accepted

that have been subject

(a) these differences

School of Medicine

described

of cell

(3).

and aggluThe method-

agglutination

by

2.5

2 2 2

2

50

60

7 70

60

Bleomycin*

Chromomycin A3

Adriamycin+

Methotrexate

1,3-Bis(2-chloroethyl60 amine)-l-nitrosourea (8tJR-I) Con A

Con A WGA WGA

Con A Con A

Con A WGA Con A WGA

Con A WGA Con A WCA

Con A Con A

100

45 3.2 6.0

150 150

75 2.5 50 2.5

120 3.5 120 3.5

200 200

Plant lectin (ug/l.2ml)

2.4

2.2 1.8 1.6

2.2 2.2

4.4 3.1 4.0 3.1

2.5 1.9 2.4 1.9

2.0 2.5

2.0

2.0 1.6 1.4

1.5 1.2

4.4 3.1 4.0 2.5

2.2 2.4 3.1 2.5

2.0 4.7

0.40

0.47

0.24 0.34 0.74

0.20 0.30

0.14 0.14 0.16 0.21 0.58

0.08 0.05 0.16 0.10

0.52 0.14 0.17 0.16

0.13 0.08

0.08 0.05 0.11 0.05

0.54 0.30 0.35 0.42

0.13 0.13

Agglutination Process Lag period (min) Rate (AA,, /2 min Control kz& Control Treated

Con A was employed in concentrations ranging from 35 to 300 ug/1.2 ml and WGA at levels of 2.5 to 6 ug/1.2 ml with each agent; for simplicity, only selected concentrations of agglutinin are listed. +The stimulatory effect of adriamycin on agglutination of Sarcoma 180 ascites cells was reported in abstract form by Murphree et al. (11). *Bleomycin was obGi=d from Bristol Laboratories in vials containing 15 units (equivalent to 8 mg) of a mixture of bleomycins with an average molecular weight of 1321.

7

2

7.5

2 7

Incubation time (h)

60

Concentration ut4

GThioguanine

Antineoplastic agent

Table 1. The Effect of Antineoplastic Agents on the Agglutination of Sarcoma 180 Ascites Cells by Concanavalin A (Con A) and Wheat Germ Agglutinin (WGA)

PreiimiMry

a characteristic

two parameters : the process

at 546 nm resulting The effects

of various

required

was secondary

relatively

to other cellular

changes in the cell by this

surface

to this

lesions;

induced by this

incubation

antibiotic

occurred

(i.e.

of differences

preparations

arabinosyl

affect

cytosine,

the rate end extent

conditions

that the effect

that the drug induced

of ultimate

cytotoxicity

agglutination

employed.

incubation

rate with cells

cells

of various

exposed

cells. those created

exposure to drug. Differin different

batches

of plant

and not listed

and S-fluorodeoxyuridine)

of agglutination

cells

BCNUand methotrexate

for 7 hr; whereas,

as 2 hr after

Other agents tested

S-fluorouracil

changes in the

of Sarcoma 180 ascites

of untreated

in the potencies

were

by Con A, which agglutinated

but at a decreased

as early

anti-

of Sarcoma 180 cells

experiments lectin

in the table

did not significantly by Con A under the

employed.

The present

findings

and those of other investigators

in the plasma membrane, due either cytotoxic

suggesting

chromomycin A3, adriamycin,

ences in the apparent rate of agglutination

and cellular

agglutination

by the purine

is conceivable

were detected

lectin-induced

and methotrexate

were the result

process

in the expression

The changes produced by chromomycin A3 required by adriamycin

are summarized in

exposure to drug using WGA. Progressive

In contrast,

enhanced the rate of plant

in absorbance

lectin-induced

(7 hr),

however, it

2.5 hr exposure to bleomycin,

agent for 7 hr.

by initiating

changes in the plasma membrane caused by bleomycin

as 2.5 hr after

normally after

the plant

of the agglutination

may be a factor

In contrast,

agent.

found as early cell

surface

agents on this process

inhibited

prolonged

created

and sedimentation.

chemotherapeutic

The alteration

of Sarcoma 180 cells. metabolite

aggregation

and bleomycin

agglutination,

warming to 37*, and a decrease

programed

from cell

6-Thioguanine

Table 1.

lag phase preceding

at 0* and allowing

1151

communicstions

amhanism of action

Aclmowledgenmt: the National

to direct

of certain

This research

(4-11)

or indirect

action,

antineoplastic

indicate

that alterations

may play a role

in the

agents.

was supported in part from USPHSGrant CA-02187 from

Caucer Institute.

References 1.

V. N. Nigam and A. Cantero,

Adv. Cancer Res. l7,

2.

A. M. C. Rapin and M. M. Burger, Adv. Cancer Res. 20,

3.

K. M. Hwang, S. A. Murphree and A. C. Sartorelli,

4.

H. B. Bosmann and D. Kessel,

5.

I. J. Fidler,

Nature 226,

1 (1973). 1 (1974).

Cancer Res. 34, 3396 (1974).

850 (1970).

P. C. Montgomery and J. P. Cesarini,

Cancer Res. 33, 3176 (1973).

1152

Preliminary Communications

6. T. E. Ukena,J. Z. Borysenko,M. J. Karnovskyand R. D. Berlin,J. Cell Biol.61, 70 (1974). 7. H. H. Yin, T. E. Upena and R. D. Berlin,Science178, 867 (1972). P. Jones and G. Robertson,Biochem.Biophys.Res. 8. A. 0. Hawtty,T. Scott-Burden, Comnun.S4, 1282 (1973). 9. S. H. Tu, R. E. Nordquistand M. J. Griffin,Biochim.Biophys.Acta 290, 92 (1972). 10.

K.

M. Hwang, S. A. Murphreeand A. C. Sartorelli,Pharmacologist l6, 210 (1974).

la, 209 (1974). 11. S. A Murphree,K. M. Hwang and A. C. Sartorelli,Pharmacologist