Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

Vol. 112, INo. 2, 1983 April

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

29, 1983

Pages 537-546

HUMAN LEUKOCYTE INTERFERON SUBTYPES HAVE DIFFERENT ANTIPROLIFERATIVE

AND ANTIVIRAL

ACTIVITIES

Fish 192, K. Banerjeel

E.N.

ON HUMAN CELLS

and N. Stebbing3

1 Division

of Infectious Diseases Research The Hospital for Sick Children 555 University Avenue, Toronto, Ont.

3Applied 1900 Oak Terrace

Received

March

16,

Institute M5G 1X8

Molecular Genetics, Inc. Lane, Thousand Oaks, Ca. 91320

1983

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-aA, B, C, D, F) and 2 molecular hybrids between them (IFN-alAD(BglI1) and IFN-aDA(BglI1)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-aB, C and F showed comparable antiproliferative activity which was greater than that of IFN-aA, D, AD(BglI1) and DA(Bgl.11). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-aA, D and AD(BglI1) were observed to be more protective than IFN-aB, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.

Cell

growth

interferon

to purified

preparations

well

as with

ranging

both effects

the different

of inhibition

2To whom requests

has been observed from

crude

IFN preparations

(11-18)

inhibitory

degrees

activity

preparations

extracts

growth

inhibitory

are

for

--in vitro are

reprints

cells

human interferon

Variations

different employed.

uncommon when normal,

should

mouse

culture-derived

and with

and --in vivo.

seen with

target not

(l-10)

tissue

with

in cell

IFN preparations Certainly hyperplastic

as

differing and

be addressed.

The abbreviations used are: IFN-a, human leukocyte interferon with the Gacid sequence of sub-types -A, -B, etc., as defined elsewhere (19,21); U/ml, international standard units/ml; WISH, human amnion cells; MDBK, bovine kidney cells; BSV-2, herpes simplex virus type 2; EMCV, encephalomyocarditis virus.

537

0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any.form reserved.

Vol. 112, No. 2, 1983

neoplastic

BIOCHEMICAL

tissue

relation

of the

between

remains

cell

same histologic

growth

origin

inhibitory

RESEARCH COMMUNICATIONS

are

activity

examined

and the

and antiviral

activity

unclear.

Cloning family

of genes

for

IFNs

of human leukocyte

subtypes

show distinct

several

viruses

IFN IFN-aAD

shows

antiviral

(21).

IFN-aAD,

purified that

various

subtypes

This degree

study

determine

whether

subtypes

correlated

undertaken

virus

hominis

order

to compare

indicate

that

antiviral

with cell

mouse cells

coat

(19).

The various

different

potency

lines

(20)

activity

(25,210.

and the hybrid

against

than

human cells

and D (22-24)and However,

which

for

activity

are

the

to date

shown antiproliferative

IFN preparations,

of a

against

on mouse cells

activity IFN-aA

two subtypes,

the existence

mixtures

lower

of the

(19,24,26).

of growth

some of the

for

reports

were

of mammalian

have generally

of buffy

(HuIFN-c0

Some antiproliferative

against

subtypes

has revealed

activites

even greater

has been demonstrated hybrid,

in bacteria

IFN subtypes

in a range

on human cells

than

AND BIOPHYSICAL

that

inhibitory

activity

the growth with using

type

2 of the

a sufficient

exert

a considerable In an attempt

activities

of the

antiviral

activities,

same cell

types

number

of different impure.

in the

properties

respective

preliminary

challenged

and encephalomyocarditis

used were

the differences

subtypes

in human cells.

inhibitory

their

2 (HSV-2)

preparations

some HuIFN-a

with virus

HuIFN-a

However,

IFN studies

herpes-

(EMCV).

In

subtypes,

various

observed

to

are

experiments not

simply

due to contaminants. MATERIALS Cell

AND METHODS

cultures

Six different human cell types were used in this study: T98G (neuroblastoma), SK-H-NA (hepatoma, provided by L. Heller from the Memorial-Sloan Kettering Cancer Research Center, New York), PLC-PR-5 (27,28) (hepatoma, expressing hepatitis B surface antigen, provided by 6. Shames and S.V. Feinman, Mount Sinai Hospital, Toronto), K562 and HL60 (2 distinct myeloid leukemic lines) and Daudi (a lymphoblastoid line, derived from B-cells). Cells are grown either as monolayer or suspension cultures at 37OC in humidified air with 5% C02, in MEM-a medium (University of Toronto tissue culture medium preparation service) su plemented with 10% fetal calf serum (Gibco), lo3 I.U. penicillin and 10 E; vg/ml streptomycin. 538

Vol. 112, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Viruses -HSV-2 stain no. 333, isolated from a primary genital lesion and provided by Dr. Carlos Lopez of the Memorial Sloan-Kettering Cancer Center,, New York, was used in this study. HSV-2 is routinely passaged ~I-38 human lung fibroblasts and EMCV on mouse L-929 cells.

on

--Interferons F:Lve HuIFN-u subtypes obtained by gene cloning in E. coli were used: IFN-rrA, B, C, D and F (19). These interferon subtypes all consist of 166 amino acid residues, except IFN-crA, which consists of 165 amino acid residues (19). The hybrid interferons IFN-aAD(BglI1) and IFN-aDA(BglI1) used consist of the N-terminal 61 or 62 residues of IFN-(rA or D respectively and the remaining C-terminal portion of IFN-aD or A, respectively (21). These recombinant IFN-as were kindly provided by Genentech, Inc., San Francisco, CA. The unpurified IFN-as B,C and F were prepared as descri ed previously and had specific (20). activities in a range from 3.2x10 k to 1.6~10~ U/mg protein Extracts of bacterial cells containing plasmids lacking IFN gene In addition, we used sequences were found to have no antiviral activity. preparations of IFN-ctA, D, AD(BglI1) and DA(Bg111) which were elec'crophoretically homogeneous and greater than 95% pure with specific activities between 0.6 and 2.2~10~ U/mg protein, assayed on MDBK cells as described elsewhere (21). A control human buffy coat IFN preparation was obtained from Dr. Norwood Hill, Wadley Institute for Molecular Medicin Houston, Texas. This natural material had a specific activity of 1x10 &, U/mg protein. In addition, lymphoblastoid IFN was sed (Wellcome Foundation, U.K.) with a specific activity of 2x10 # U/mg protein. IFN titers were determined on WISH or MDBK cells challenged with VSV using a 50% cytopathic end-point and converted to international units using an NIH standard (G023-901-527) included in each assay. Rabbit anti-leukocyte IFN was obtained from Interferon Sciences, Inc. New Brunswick, NJ. In Vitro

Assay

for

Antiproliferative

Activity

Cells were seeded in individual wells of 96-well Microtest II tissue Ca.) at a density of 103 cells/well culture plates (Falcon, Oxnard, (5x10:$/ml) for the monolayer cultures PPL-PR-5, SK-H-NA and T98G, and at 2x1.03 cells/well (104/ml) for the suspension cultures K562, Daudi and HL60. These are the optimal seeding concentrations to avoid early confluence of cultures. The growth inhibitory activities of the various interferons were determined by inoculating cultures at the times of their initiation with dilutions of standardized doses of the IFNs. At predetermined times, monolayer cells were ethanol fixed, crystal violet (0.1% in 2% ethanol) stained and inhibition of growth estimated from absorbance at 570 nm using a Microplate Reader MR600 and a calibration of absorbance against cell numbers. Suspension culture cell aliquots were counted directly in a Coulter Counter (model ZBl). The growth inhibitory activity of the various IFN-cts is expressed as a percent of control growth. For the monolayer cultures, the absorbance at 570 nm of cells not treated with IFNs and stained with For cells fixed crystal violet served as a standard for normal growth. and st.ained during their logarithmic phase of growth, their absorbance at 570 nm is directly proportional to the number of cells present, for all IFN-treated cells were scored for percent the monolayer cell types used. of control growth by comparing their absorbances at 570 nm with corresIFN-treated suspension cultures were ponding cell standards. Similarly, scored for percent of control growth by comparing their cell numbers direct.ly with cell standards. 539

Vol. 112, No. 2, 1983

In Vitro

Assay

BIOCHEMICAL

for

Antiviral

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Activity

Tq8G cells were seeded in individual wells of 96-well Microtest II tissue culture plates and pretreated with dilutions of the respective IFN preparations for 24 hrs. At the time of t+us inoculation the IFNs were (determined on Vero removed and either 3 ~1 HSV-2, TCID50 lo- 1 cells) or lo4 PFU EMCV added to individual wells in 100 ~1 medium. To terminate the experiment cells were fixed with 95% ethanol. The extent of HSV-2 and EMCV infection was determined by spectrophotometric fixed cells were crystal violet estimation of viral cytopathic effect: stained and the inhibition of virus infection estimated from absorbance at 570 nm. Cells were Antiviral assays were also set up using K562 cells. seeded in wells of the Microtest culture plates and pretreated with the respective IFN preparations for 24 hrs. The IFNs were then removed and either HSV-2 or EMCV added as above in 100 ~1 medium. Viral adsorption was permitted for 2 hrs after which time the medium was replaced with fresh medium. Cells were incubated for 23 hrs then assayed for virus yield by quantitating viral CPE on Vero monkey kidney cells. RESULTS Antiproliferative

Activities

The growth

inhibitory

on human monolayer experiments

IFN-olB,

inhibition

IFN-aA

different

cell

was completely IFN-aDA extent the

trations

by

extent,

cell

types

is

50% inhibition the

growth

was inhibited and PLC-PR-5

At 103 U/ml,

these

IFN-us

IFN-es

were

Overall

the The

IFN preparations

for

types

IFNs inhibited

as concen-

B, C and F, at except growth

SK-H-NA, to a lesser

and DA (BglII), HL60,

more sensitive

A, D and AD (BglII) 540

of the

activity.

The IFN-crs

by O-25% on K562,

the

Where growth

I expressed

A, D, AD (BglII)

lines

patterns

antigrowth

the cell

with

and

IFN treatments.

in Table

all

line,

For the

IFNs:

of

and growth

least

of growth.

growth

observed

IFN-aAD(BglI1)

by the various

summarized

The

pronounced

degenerated.

the

examined

1 and 2 show typical

inhibition

with

eventually

induced

For the SK-H-NA

The Daudi

hybrids

demonstrated

inhibition

the

were

was measured.

demonstrated

observed

cells

by 2-19s.

103 U/ml,

these

hybrid

Figures

in morphology

not

suppressed

inhibited

6Q-70%.

cells.

were

IFN-es

growth

exceeded

and the

types

causing

103 U/ml,

that

changes

of growth

different

growth

Gross

(BglII)

of cell

and IFN-crF

and IFN-aD

IFN-aDA(Bg1).

cultures.

inhibition

IFN-(rC

of cell

subtypes

of the various

and suspension

in which

subtypes

effects

at

T98G and SK-H-NA to certain inhibited

growth

of

BIOCHEMICAL

Vol. 112, No. 2, 1983

IFN

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

U/ml

I?igure

1

Growth inhibitory activities of HuIFN-as on human suspension cultures. C, DAUDI; A, K562; &, HL60; Cells (10 /ml) were incubated in MEM-u medium containing 10% fetal calf serum at 37oC in the presence of an IFN for 98 hrs (~5621, 118 hrs (HL60) and 96 hrs (DAUDI). Cell numbers were counted in a Coulter Counter. The values are the average of replicate samples each. IFN-aA ( 0 1; IFN-aB ( 0 ); IFN-aC ( . ); IFN-aD ( 0 1; IFN-aF ( 0 ); IFN-aAD(BglI1) ( v ); IFN-aDA(BglI1) ( l ).

Figure

2

Growth inhibitory activities of HuIFN-as on human monolayer cultures. A, PLC-PR-5' B, SK-H-NA; C, T98G; Cells (5x10 1 /ml) were incubated in MEM-a medium containing 10% fetal calf serum at 37OC in the presence of an IFN for hrs (PLC-PR-5), 125 hrs (SK-H-NA) and 112 hrs (T98C). Cell numbers were estimated by spectrophotometric determination. The values are the average of replicate samples each. IFN-aA ( 0 ); IFN-aB ( 0 ); IFN-aC ( A ); IFN-aD ( 0 ); IFN-uF ( 0 ); IFN-aAD(BglI1) ( v 1; IFN-aDA(BglII) ( l 1; buffy coat IFN ( n ).

541

144

BIOCHEMICAL

Vol. 112, No. 2, 1983 of Daudi growth

cells

by 42-522.

of PLC-PR-5

whereas

Daudi

of the range To test

by 52%.

are

most

of IFN-as whether

Overall

these

sensitive

tested,

in the crude

and -aF may influence

the

inhibitory

the

activity

L-cells.

Any bacterial

preparation inhibit

that

might

the growth

of IFN-aC

extract

growth

IFN-aAD(BglI1)

at 20 and 500 U/ml

approximately

30 and 65% respectively,

had no inhibitory of bacterial were

found

interferon

effect cells

specific

preparation

proliferative Antiviral

effects

-aC IFNs

on mouse IFN-aC

likewise

preparation

growth

of

of L-cells

at both

by

concentrations Furthermore,

lacking

inhibitory

IFN-aB,

should

of L-cells.

to have no growth

sensitive.

in the crude

a pure the

effects

of these

present

IFN-aC

plasmids

antibody

least

was determined

inhibited

on the growth

containing

are

preparations

Whereas

that

inhibitory

of human cells

of mouse cells.

inhibited

indicate

activities

impurities

inhibit

results

cells

impurities

antiproliferative

AD (BglII)

to the growth

the SK-H-NA

growth

RESEARCH COMMUNlCATlONS

at 103 U/ml,

Similarly,

cells

cells

AND BIOPHYSICAL

interferon

activity,

extracts

gene sequences

and a human leukocyte

completely

abrogated

the anti-

of IFN-aC.

Activities

The antiviral

effects

T98G and K562 cells

of the

challenged

interferon

with

subtypes

were

examined

The data,

HSV-2 and EMCV.

on

shown

in

TABLE I GROWTH INHIBITORY

ACTIVITIES

OF HuIFN-ns

ON DIFFERENT

HUMAN CELL

%oa CELL

TYPE

T98G

IFN-fiA

IFN-afl

IFI-ac

ZlU?

PLC-PR-5

2x10:

SK-H-NA

.5x104

DAUDI

7.2x102 l.L5x103

6.25~10~

.10x

13

a IFN concentration, - not

,103

3.5x102

4.3x10'

different appropriate between U/ml,

70 3X10:'

.l.L5x103

3.7x102

.lOj

50% growth

BUFF-f COAT IFN

LYMPHOBLASTOID IFN

.103

'5XlOL .I03

.103

IFN subtypes and hybrids cell type. Replicate days are represented

causing

It?-'XDA (Bgl II:

8.7x10;

.10x

5.9x102

4-6

IF?-IJM (Bgl II)

6.7~10'

1.4x10"

15.6

HL60

of the with the assayed

5.2x102

IM-aF

6.5~10~

K562

Dilutions seeding cultures

IFN-nD

'TYPES

were assays above.

inhibiclon

done

542

)1.25X103

,1.25x103

.I03 introduced were set

.5x104 '103

,103 into up in

individual all cases

9x102 '1.25X10' ‘103 wells at the time and the mean values

of of

BIOCHEMKAL

Vol. 112, No. 2, 1983

Figure

2, demonstrate

different

that

effects

(BglII)

are

IFN-uF.

is

in the

Protection

from i.e.

the

both

reduced

by 85-98%.

various

IFN preparations

The extent

with

of the

relative IFN-uD

IFN-aB,

antiproliand IFN-uAD

IFN-aC

the IFN-as

in K562 cells,

of antiviral

against

in Table

their

subtypes

infections

virus

activities

IFN-aA,

was more pronounced

against

summarized

than

RESEARCH COMMUNICATIONS

antiviral

distinct

same cells,

more antiviral

(BglII)

is

the relative

IFN preparations

ferative

AND BIOPHYSICAL

and

A, D and AD

where

activity

viral

induced

CPE was

by the

HSV-2 and EMCV infections

of K562 cells

II.

~~ISCUSSION --__ The relative EuIFN-as

remained rate

erowth

inhibitory than

growth

inhibitory

the

same on all

was severely activities

IFN-aA,

D, AD(BglII),

cell

lines.

between

IFN-aDA(BglI1)

IFN-eA

and D or the indicating

It

titers

reverse

types

the cells

be

neither

of the

IFN-crB

IF&NC

and greater

coat

were

used in

differences

the

parental

The

and lymphoblastoid the

in efficacy

IFN subtypes, supports

N- or C-terminal

TABLE ACTIVITIES

comparable

IFN-aAD(BglI1)

the

ANTIVIKAL

that

Where the died.

interferons

noted

hybrid,

tested.

eventually

and buffy

of the

and either

that

cell

DA(BglI1)

should

of each of the cloned

C and F were

of IFN-crB,

various

results

the

suppressed

when similar

interferon,

activities

previous

portions

of the

II

OF HuIFN-as

ON K562

CELLS

WOa VLiWS

IFN-mA

E?Ii:V

1.75x10'

HS7'-2

1.45x102

a

5x102

-

G62 cells were I-hen challenged ;Issayed on Vera

pre-treated with either cells.

FN cont:entration,

U/ml,

>103

IFN-rtD

IF'N-uF

2x10'

>103

70

>103

'103

37

>103

dilutions of After 23 hours

the different virus yield

2.5x102

for 24 hrs. with EMCV or HSV-2. causing

50% CPE on Vera

543

cells

IFN-WG (Bgl II)

IFNkXDA (Bgl II)

IFN-;rs was

Vol. 112, No. 2, 1983

Figure

alone

IFN-aA, antiviral

IFN-aC

leukocyte

IFN subtypes

degree inhibitory growth

RESEARCH COMMUNICATIONS

selection

not

all

receptor

binding

were

observed

to be more effective

cells

challenged

These

used

for

cell

with

results

that

biological

antigrowth

as

HSV-2 and EMCV than

suggest

have distinct as either

(Zq,jO).

the

various

properties or antiviral

that agents

3).

Although purified

against or IFN-(rF.

useful

(Figure

determine

D and AD(Bg111) agents

IFN-aB,

AND BIOPHYSICAL

3 Antiviral activities of HuIFN-as on human cells. A, K562 + HSV-2; B, K562 + EMCV; C, T98G + HSV-2; D, T98G + EMCV; Cells were pre-treated with an IFN for 24 hrs then challenged with either HSV-2 or EMCV. For A and B virus yield was determined by resultant CPE on Vero cells, and for C and D viral CPE was quantitated directly by spectrophotometric estimation. The values are the average of replicate samples each. IF&crA ( 0 ); IFN-aB ( 0 ); IFN-aC ( * ); IFN-aD ( 0 1; IFN-aF ( 0 ); IFN-aAD(BglI1) ( v ); IFN-aDA(BglI1) ( + 1.

interferons

prove

BIOCHEMICAL

the

studies

IFN preparations, of purification activity inhibitory

the since

at all, activity

reported results

a crude

here do not

bacterial

and an antibody of a crude 544

included

seem to depend extract

preparation

preparation

use of highly on the

had no growth abrogated

of IFN-UC.

The

the

may

BIOCHEMICAL

Vol. 112, No. 2, 1983

interfceron

antibody

bacterial

Whether

IFNs might

differmential

is

not

that

particular

those

antiproliferative

with

of new hybrid produce agents

This

into

I whereas

effects to include

(32).

range

this

idea

of cell further

from

particular

respective

parental

IFN

and an intermediate to be found

at

154 and 160 (32). into

structural in antiviral

studies. types

and

By extending

and viruses,

and identify

it

IFN subtypes

or antiviral

activity.

parental

subtypes

of IFNs more potent

for

leukocyte

the

in the present

there

of a

C and F fall

Thus,

The

implications

the differences

antiproliferative

interferons

the

a wider

83,

IFN-aB,

for

observed

to test

optimum

basis

79,

that

IFN subtypes

residues

71, 78,

group

may be the

important

groups

have

activity

effects

cloned

two major

16,

might

(20,26,31).

suggests

derived

acid

that

antiproliferative

The various

amino

uncertain.

replication

may have

into

group

a new generation than

against

of viral

pathways

and antiviral

result

14,

remains

activity

of recombinant-DNA

namely

be possible

hybrids

antigrowth

or the intermediate cited

or virus

antiviral

on alternative

IFN-crA and D fall

studies

in rabbits

and inhibition

the greatest

indications.

positions,

differences

any of the

was raised

biochemical

growth

been classified

depending

II,

antibody

different

the

selection

have

defined

neutralize

same mechanisms

the greatest

clinical

subtypes

should

by the

IFNs with

between

the

particular

these

multiplication

IFN subtype.

regarding

group

of cell

on cell

demonstrate

no relation

group,

the

induce

effects

observation

not

because

are mediated

Various

should

RESEARCH COMMUNICATIONS

IFN.

inhibition

replication

did

preparation

contaminants

?uman leukocyte

AND BIOPHYSICAL

as antigrowth

Synthesis could

thus

or antiviral

subtypes.

ACKNOWLEDGMENTS We thank Drs. B.R.G. Williams and S. Read for their support and helpful discussions in the preparation of this manuscript. This work was supported in part by a grant made available to E.N. Fish by the Canadian National Cancer Institute. --REFERE:NCES 1.

Buffet, Cancer

K.F., Ito, M., Inst. -60:243-246.

Cairo,

A.M.,

Carter,

W.A.

(1978)

J. Natl.

or

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4. 5. 6. 7. 8. 9.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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