Differential effect of recombinant human leukocyte interferon on human leukemic and normal myeloid progenitor cells

Vol. 108, No. 3, 1982 BIOCHEMICAL AND BlOPHYSiCAL RESEARCH COMMUNICATIONS October 15, 1982 Pages 1048-1055

DIFFERENTIAL EFFECT OF RECOMBINANT HUMAN LEUKOCYTE INTERFERON ON HUMAN LEUKEMIC AND NORMAL MYELOID PROGENITOR CELLS4

Steven Grant1$3*5, Kapil Bhallaly3,

I. Bernard Weinsteinly3, Sidney

Pestka4 and Paul B. Fisher2y3

Departments of Medicine1 and Microbiology* and Cancer Center/Institute of Cancer Research,3

Columbia University, College of Physicians and Surgeons, New York, New York 10032 and Roche

Institute of Molecular Biology4, Nutley, New Jersey 07110

Received August 31, 1982

SUMMARY: Two separate clones of recombinant leukocyte interferon (IFLrA and IFLrD) inhibited the cloning efficiency in soft agar of the human leukemia cell lines HL-60 and KG-l. Inhibition of the growth in agar of normal human bone marrow myeloid progenitors was also observed, but this required consider- ably higher concentrations. IFLrA and IFLrD also inhibited the growth of HL-60 and KG-l cells in suspension culture. This antiproliferative effect did not appear to be due to induction of maturation of these cells. Our results suggest that homogeneous preparations of interferon may be capable of exerting selective antiproliferative effects on malignant human myeloid progenitor cells in comparison to their normal counterparts.

INTRODUCTION

Interferons (IFS) are a family of proteins with antiviral activity produced

by animals and cells in culture in response to viruses, as well as certain non-

viral substances (l-4). In addition to their antiviral effects, they exhibit a

wide variety of other effects, including inhibition of the growth of both normal

and transformed cells (5-6). Clinical studies utilizing partially-purified human

leukocyte interferon have been associated with the development of neutropenia, sug-

gesting that these preparations of interferon were toxic to normal myeloid progenitor

cells (7). These findings are consistent with in vitro studies which demonstrated

5. Fellow of the Leukemia Society of America to whom reprint requests should be made.

Abbreviations used: TPA, 12-O-tetradecanoyl-phorbol-13 acetate; DMSO,

dimethyl sulfoxide

0006-291X/82/191048-08$01.00/0 Copyright 0 I982 by Academic Press, Inc. All rights of reproducfion in any form reserved. 1048

Vol. 108, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that certain interferon preparations inhibited the formation of normal human

granulocyte macrophage colonies (CFU-GM) in soft agar (8,9). To date, normal

and malignant cells have exhibited varying degrees of sensitivity to the in vitro

growth inhibitory effects of the various interferons (10).

After the isolation (11) and expression (12) of DNA recombinants contain-

ing sequences of human leukocyte interferon, homogeneous preparations of human

leukocyte interferon were prepared (13). We have studied the inhibitory effects

of two types of human leukocyte interferon produced in Escherichia coli (IFLrA

and IFLrD) on the cloning efficiency of normal human bone marrow progenitor

cells in soft agar. In addition, we have examined the effects of these inter-

ferons on the in vitro proliferative capacity of two human leukemic cell lines,

HL-60 and KG-l. These cell lines have been the focus of considerable interest

in view of their capacity to undergo terminal differentiation in the presence

of a variety of compounds, including dimethyl sulfoxide (DMSO) or phorbol esters

(14,15). The purpose of the present study was to determine whether these inter-

ferons might exert a preferential inhibitory effect on the proliferation of leu-

kemic versus normal human myeloid progenitor cells in soft agar. A second aim

of this study was to determine whether the growth inhibitory effects of IFLrA or

IFLrD on HL-60 and KG-1 cellswereassociated with morphologic or functional

evidence of differentiation.

METHODS

HL-60 and KG-1 cells are derived from the original lines initially described (14,15). They were maintained in RPM1 medium supplemented with 1% sodium pyruvate and nonessential amino acids and 10% heat-inactivated fetal calf serum (GIBCO). Cells were subcultured twice weekly in 75 cm2 sterile plastic tissue culture flasks (Corning) and kept in a fully-humidified 37"C, 5% CO2 water- jacketed incubator (Napco). Recombinant interferons (IFLrA and IFLrD) were prepared as described (13). The interferon preparations were stored in sterile 2-ml vials at -8O"c, thawed immediately prior to use and diluted to the appropriate concentration in RPM1 medium. TPA (12-O-tetradecanoyl-phorbol-13-acetate) and DMSO were purchased from Sigma Chemicals, St. Louis, MO. In suspension culture growth experiments, 5 ml of HL-60 and KG-1 were seeded into 35mmsix-wellplates (Costar) at an initial cell density of lo5 cells/ml. TPA (lo-7M), DMSO (1.25%) or recombinant interferon (IFLrA or IFLrD, 200-5.000 I.U./ml) were added to the wells, and the plates were placed in the 37OC, 5% CO2, fully-humidified incubator. Aliquots of 0.5 ml were withdrawn at 24-hr intervals, and cell density determinations obtained with a model ZBI Coulter counter (Hialeah, Fla.). At the end of 5 d, the cells were pelleted and cytocentrifuge preparations made with a Shandon cytocentrifuge. After air drying, the slides were stained with Wright-Giemsa, and a differential count performed by light microscopic examination of at least 200 cells. For nitro-blue tetrazolium

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dye reduction studies, 1 ml of cells suspended at 2 x 106 cells/ml in RPM1 medium supplemented with 20% FCS was incubated for 20 min at 37OC with equal volume of 0.2% NBT (Sigma) in the presence of 200 ng of TPA. The percent of cells containing intracellular reduced blue-black formazan deposits was then determined on Wright-Giemsa stains of cytospin preparations of incubated cells.

The ability of HL-60 and KG-l cells to form colonies in soft agar in the presence of varying concentrations of interferon was determined by a slight modification of a previously described technique (16). Cells were plated in 18 mm 12-well dishes (Costar) utilizing a two-layer agar system. The bottom layer consisted of 0.5 ml of RPM1 medium supplemented with 1% sodium pyruvate, 1% nonessential amino acids, 20% (V/V) fetal calf serum and 0.5% bacto agar (Difco). The top layer containing the cells consisted of 0.5 ml of RPM1 medium, 20% (V/V) fetal calf serum, 0.3% bacto agar, and the appropriate concentration of ILFrA or IFLrD. HL-60 cells (2 x 103) or KG-l cells (5 x 103) were plated for each condition. After the agar hardened, 0.1 ml of GCT-conditioned medium (GIBCO) was added to each plate as a source of colony-stimulating activity (17). The plates were then placed in a 37OC. 5% C02, fully-humidified incubator for 10 d. At the end of this period, the plates were removed, and the number of colonies, consisting of groups of 50 or more cells, scored with the aid of an Olympus Model CK inverted microscope. Inhibition of colony growth was expressed as (l-No/NC) x 100, where No equals control colony growth and NC equals the number of colonies formed in the presence of a particular concentration of interferon. The I.C.50 was defined as the interferon concentration which resulted in a 50% reduction in-colony growth when compared to controls.

The effect of IFLrA and IFLrD on the growth in soft agar of normal human bone marrow progenitor cells (CFU-GM) was determined by a minor modification of a previously described method (18). Bone marrow cells were obtained with informed consent from patients undergoing routine diagnostic aspirations who did not have a hematologic malignancy. These studies have been sanctioned by the Investiga- tional Review Board of the Columbia University College of Physicians and Surgeons. The cells were passed through a 26-gauge needle to disperse clumps, and diluted 1:4 with McCoy's 5a medium (GIBCO) supplemented with sodium pyruvate, essential and nonessential amino acids, MEM vitamins, asparagine, glutamine and serine. The suspension was then gently layered over 5 ml of lymphocyte separation medium (sp. grav. =1.077-1.081; Bionetics, Kensington, Md) in sterile 20-ml centrifuge tubes and centrifuged at 400 xg for 38 min. The interface layer, containing the mononuclear cells, was extracted with a sterile Pasteur pipette, washed three times with supplemented McCoy's 5a medium and cell counts obtained with a hematocytometer. The method for determining the effect of IFLrA or IFLrD on the growth of bone marrow cells in soft agar was the same as that described for HL-60 and KG-l cells, except the supplemented McCoy's 5a medium was used in place of RPM1 and 105 bone marrow cells were plated in each well, After 10 d of incubation, the number of colonies, consisting of groups of 50 or more granulocytic or macrophage-like cells, was scored with the aid of an Olympus inverted microscope. Inhibition of normal bone marrow myeloid progenitor cell growth in soft agar by IFLrA or IFLrD was expressed in the same manner as for HL-60 and KG-l cells.

RESULTS

Figure 1 illustrates the effects of IFLrA and IFLrD on inhibition of

colony formation by KG-l, HL-60 and normal human bone marrow progenitors in

soft agar. The inhibitory effects of both types of interferon was greater for

HL-60 and KG-1 cells than for normal marrow cells over the entire range of inter-

feron concentrations tested (200-5,000 I.U./ml). At high IFLrA concentrations

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20-i

I,, , , , ( 1000 3000 5000

IFLr DOSE (units / ml1

Figure 1: -- The effect of IFLrA and IFLrD on the cloning efficiency of HL-60, KG-l and normal bone marrow cells.

The inhibitory effect of IFLrA (A) and IFLrD (B) on the cloning efficiency of HL-60 (a), KG-l (A) and normal human bone marrow porgenitor cells (O-O) was assessed as described in Material and Methods. 5,000 I.U./ml is equivalent to 2.8 and 165.0 x lo4 pg protein/ml for IFLrA and IFLrD, respectively.

(5,000 I-U./ml), inhibition of KG-l and HL-60 cell colony formation was about 98

and 94%, respectively, but only 62% for normal marrow cells. With all three cell

types, the inhibitory effects of IFLrD were less than those obtained with equal

concentrations of IFLrA. An IFLrD concentration of 5,000 I.U./ml was associated

with about 85 and 83% reduction in HL-60 and KG-l cell colony formation, but

only a 48% reduction in colony formation by normal human marrow cells. With

IFLrA, the I.C.50 (i.e., the concentration which produced a 50% reduction in

colony formation) was 400 and 300 I.U./ml for HL-60 and KG-l cells, respectively,

and averaged 1,250 I.U./ml for the seven nonleukemic bone marrow samples. With

IFLrD, the I.C.50 was 650 and 950 I.U./ml for HL-60 and KG-l cells, respectively;

the average inhibitory effect of an IFLrD concentration of 5,000 I.U./ml in four

bone marrow samples was slightly less than 50%. In the seven normal bone marrow

samples exposed to IFLrA, the I.C.50 ranged from 850 to 1,900 I.U./ml (data not

shown). In only one of the four bone marrowsamples exposed to IFLrDdid aninterferon

concentration of 5,000 I.U./ml result in greater than a 50% reduction in colony

formation (53%).

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The inhibitory effects of IFLrA and IFLrD on the growth of HL-60 and KG-l

cells in suspension cultures are given in Table 1. A 4-d exposure to 5,000

I.U./ml of IFLrA or IFLrD resulted in about 49 and 35% inhibition of cell

growth, respectively, for both cell types. Interferon concentrations of

200 I.U./ml or less had negligible effects on cellular proliferations (not

shown). Thus, as for colony growth in soft agar, IFLrA was more inhibitory

to cell growth than IFLrD at each interferon dose tested (~(0.05). The

growth inhibitory effects of IFLrA of IFLrD were not associated with evidence

of morphologic or functional induction of differentiation in either HL-60 or

KG-~ cells, even at the highest concentration tested, i.e., 5,000 I.U./ml. In

contrast, the majority of HL-60 and KG-1 cells exposed to 1.25% DMSO or lo-TM

TPA matured past the promyelocyte stage or became capable of reducing NBT dye

(data not shown here).

DISCUSSION

These results demonstrate that two different homogeneous preparations of

interferon produced in bacteria by recombinant DNA techniques are inhibitory to

the in vitro growth of both leukemic and normal myeloid progenitor cells. It is

of particular interest that we found that both of these highly-purified inter-

ferons preferentially inhibited the leukemic cell cultures, since in previous

studies with partially-purified myeloid, lymphoblastoid or fibroblast interferons,

there appeared to be equal inhibition of the growth in soft agar of leukemic and

normal myeloid cells (19). It is conceivable that impurities present in previous-

ly available interferon preparations may be responsible for the lack of a selective

effect on leukemic cells, although this hypothesis requires further testing.

Little is known about the mechanisms of regulation of cell growth and dif-

ferentiation of interferons. Studies by Verma et al have suggested that par-

tially-purified human leukocyte interferons are capable of blocking the differen-

tiation of granulocytic progenitor cells (8,9). In a recent study, highly-puri-

fied human leukocyte interferon did not induce differentiation in HL-60 cells (20),

nor did we see such an effect with IFLrA or IFLrD. On the other hand, syner-

gistic effects on differentiation were noted when interferon was combined with

1052

Table

1:

Ef

fect

of

in

terfe

rons

on

th

e gr

owth

an

d di

ffere

ntia

tion

of

susp

ensio

n cu

lture

s of

HL

-60

and

KG-l

cells

.

COND

ITIO

NS

CONT

ROL

5000

IF

LrA

(I.U

./ml)

2000

10

00

500

5000

IF

T.rD

(I.

lJ./m

l>

2000

10

00

500

1.

Gro

wth

(X

inhi

bitio

n at

96

hr

) a.

HL

-60

0 49

.4k4

.2

44.3

?3.8

35

.723

.1

17.3

t2.4

35

.7k3.2

31

.924

.2

27.O

t2.8

8.

321.

6

b.

KG-l

0 47

.Ok3

.8 37

.6~4

.1

33.2

t2.9

15

.651

.8

35.6

23.1

24

.853

.1

17.9

22.9

6.

3t1.

4

2.

Mor

pholo

gic

mat

urat

ion

afte

r 96

hr

(X

m

yelo

cyte

s,

met

amye

locy

tes,

ba

nds,

se

gmen

ts)

a.

HL.-6

0 5

4 1

3 N

.D.

5 2

4 N

.D.

b.

KG-1

3

2 3

1 N

.D.

3 2

4 N

.D.

3.

% N

BT

posi

tive

cells

(9

6 hr

) a.

HL

-60

3 2

2 1

N.D

. 3

2 2

N.D

.

b.

KG-l

3 3

1 2

N.D

. 3

2 2

M.D

.

Cells

we

re

seed

ed

at

5 x

lo4

cells

/ml

in tis

sue

cultu

re

flask

s co

ntai

ning

th

e de

signa

ted

conc

entra

tions

of

in

terfe

ron

and

cell

conc

entra

tions

de

term

ined

at

da

y 4.

G

rowt

h in

hibi

tion

in in

terfe

ron-

treat

ed

cells

wa

s ex

pres

sed

as

a pe

rcen

tage

re

lativ

e to

un

treat

ed

cont

rol

cells

. Va

lues

re

pres

ent

the

mea

ns

for

four

se

para

te

expe

rimen

ts

+ 1

S.D

. Th

e ce

ll de

nsity

in

cont

rol

cultu

res

at

the

end

of

96

hr

was

4.0

?r 0

.5

x lo5

ce

lls/m

l. At

th

e sa

me

time

cells

we

re

eval

uate

d fo

r ev

iden

ce

of

mor

phol

ogic

or

func

tiona

l ev

iden

ce

of

mat

urat

ion

(see

"M

ater

ials

and

Met

hods

").

N.D

. =

not

done

.


inducers such as TPA or retinoic acid (20). Thus, the antiproliferative effects

of interferon are not always simply due to induction of terminal differentiation,

although the interferons can under certain cases influence differentiation. It

has been found that a variety of chemotherapeutic agents, particularly those

that interfered with DNA synthesis, can under certain conditions induce HL-60

cells to develop into more mature forms (21). However, these same agents exhi-

bit only cytotoxic effects, without inducing defferentiation, when administered

at significantly higher concentrations. One might speculate, therefore, that

agents which inhibit the proliferation of malignant cells may render these cells

more sensitive to factors capable of enhancing differentiation. If this hypo-

thesis is correct, efforts to combine interferons with antimetabolites or speci-

fic inducers of leukemic cell differentiation might prove to be an effective

strategy in cancer therapy.

Recent clinical trials with impure preparations of human leukocyte inter-

feron in the treatment of hematologic malignancies have shown that myelosuppres-

sion is the dose-limiting factor with respect ot toxicity (22). It is possible

that recombinant interferon preparations might have less of a myelosuppressive

effect. Indeed, preliminary clinical trials with IFLrA suggest that this form

of interferon may cause less bone marrow toxicity than previously available prepa-

rations (23,24). These studies demonstrated that peak serum interferon levels as

high as 1,000 I.U./ml were achievable in some patients so treated, and that peak

levels of 500 I.U./ml were possible in the majority of patients (24). Thus,

further laboratory and clinical studies with homogeneous preparations of inter-

feron, alone or in combination with other agents, may lead to more effective therapy

for certain malignancies,

ACKNOWLEDGEMENTS: This work was supported in part by the National Cancer

Institute (Award CA-13696), the Pharmaceutical Manufacturers Association, the

William J. Matheson Foundation, the Cele Butwin Foundation and Hoffman-LaRoche.

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Differential effect of recombinant human leukocyte interferon on human leukemic and normal myeloid progenitor cells