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J. gen. ViroL 0976), 33, 351-354
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The Production and Action of Interferon in Chinese Hamster Cells
(Accepted 30 June I976)
SUMMARY
The interferon system has been investigated in primary cell cultures established
from Chinese hamster embryos and new born pups. Interferon synthesis was
induced with Sindbis virus, ultraviolet irradiated Newcastle disease virus (u.v.NDV) and with polyriboinosinic acid-polyribocytidyIic acid complex [poly (rI).
poly (rC)]. Only u.v.-NDV induced significant production of interferon, maximum
amounts being produced in 'aged' ceils. Its apparent tool. wt. was 25 ooo. C H O - K t
cells, an established line of Chinese hamster cells, did not synthesize interferon in
response to viruses, but were sensitive to its action.
A functioning interferon system has been discovered in all classes of vertebrates and
many mammalian species (Ho, I973 ). Cells derived from the Chinese hamster (Cricetulus
griseus) have been increasingly utilized in biological research because of advantages they
offer over other cells. They have a small number (22) of easily identifiable chromosomes and
grow rapidly in culture with a generation time of twelve hours, and stable long term cultures
are readily obtainable. They have been used, for example, in life-cycle analysis (Petersen,
Tobey & Anderson, I969), biochemical genetics (Patterson, I975), and somatic cell genetics
(Kao & Puck, I972 ). In virology, they have been used to study cytolytic viruses (Stampfer,
Baltimore & Huang, I969) and tumour viruses (Lehman & Defendi, I97o). As far as one
can ascertain there are no reports in the literature that Chinese hamster cells possess a
functioning interferon system, and indeed there are indications to the contrary (D. Burke,
personal communication). In this study I show that primary cultures of Chinese hamster
cells synthesize interferon and respond to it.
Primary cell cultures were prepared by standard techniques from Chinese hamster
embryos and new born pups (lung, kidney, heart, iris and skin). Cells were plated in growth
medium [Ham's FI2 supplemented with 8 ~ foetal bovine serum, penicillin (ioo units/ml)
and streptomycin (5o #g/ml)] at approx. 4 x io 5 cells/cm 2 in Ioo mm Petri dishes (Flow
Laboratories) and confluent monolayers were established within one week. These were
infected with Sindbis virus at 2o to ~oo p.f.u./cell or u.v. inactivated Newcastle disease virus
(u.v.-NDV) at 2oo to IOOO particles/cell (Morgan, Colby & Hulse, I973), or were treated
with poly (rI).poly (rC) (P.L. Biochemicals Inc.) at Ioo #g/ml in the presence or absence
of diethylamino ethyl (DEAE) dextran (Pharmacia) at IOO #g/ml for I h at 37 °C. After
virus adsorption or poly (rI).poly (rC) treatment the cells were washed with medium and
incubated overnight at 37 °C with Io ml fresh growth medium. The culture fluids were
harvested, centrifuged to remove cell debris, cooled to 4 °C and made o'I5 M with respect
to perchloric acid. After 7z h at 4 °C, the precipitate was removed and the supernatant fluid
neutralized with KOH. Potassium perchlorate was removed by centrifugation and the
resultant preparation stored at - 7 o °C. The material has remained stable for over six
months. Interferon activity was measured by the dye-uptake method (Finter, I969). One to
two x lO5 cells were added to the wells of a I o o m m 2 dish (2 5 compartments/dish;
Sterilin: 3o6V) and allowed to attach overnight. Dilutions of interferon in I ml of growth
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352
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medium were then applied and incubated for I8 h at 37 °C. The fluids were removed and
the monolayers washed. Sindbis virus, so p.f.u./cell, was adsorbed for 30 rain and then a
further I ml of growth medium was added. The cells were incubated for a further 36 h when
neutral red uptake was determined colorimetrically. The end point was the dilution at which
an interferon preparation gave rise to dye uptake mid way between that in the virus control
and uninfected cell control. The reciprocal of this dilution gave the titre of the preparation
in 50 ~ dye-uptake DUs0/ml. Samples (I ml) of a Chinese hamster embryo cell interferon
reference preparation were stored at - 7o °C and dilutions of this preparation were included
in each set of assays.
Neither Sindbis virus nor poly (rI). poly (rC) led to the appearance of detectable amounts
of antivirus activity in the medium of treated cells, although treatment with the latter did
render all cells resistant to some extent to challenge with Sindbis virus. In contrast, u.v.NDV did give rise to material with antivirus activity, especially when the cells used had
been 'aged' in vitro for ten days after reaching confluency (Carver & Marcus, 2967; Table
0. Although the material has not yet been purified it has been shown to have a number o f
the properties characteristic of interferons (Lockhart, I966 ). It is non-cytotoxic, and stable
at pH z.o and 4 °C and 37 °C for at least I h but not at 60 °C for I h. Treatment with
trypsin (Worthington Biochemicals) at 5o/zg/ml for I h at 37 °C abolished biological activity
whereas treatment with pancreatic ribonuclease (Worthington Biochemicals) at 8o/~g/ml
did not. The material is active on Chinese hamster cells but not on human cells (WI-38 and
primary foreskin fibroblasts). It is of interest that a preparation of a standard human
leukocyte interferon (National Institutes of Health, reference reagent G-o23-9oi-527;
2oooo units]ml) was active on Chinese hamster cells (only 5-to Io-fold less active than on
human foreskin fibroblasts). The antiviral activity of human leukocyte interferon on a
number of heterologous cells has been previously reported (Gresser et al. I974). Chinese
hamster interferon protected Chinese hamster cells from the following viruses: Sindbis,
Semliki Forest, Mengo, encephalomyocarditis and vaccinia. The tool. wt. of the active
species was determined by gel-filtration chromatography (Andrews, I964) using Sephadex
G-I5o with an eluant of phosphate buffered saline (o.I 4 M-NaCI, o.I M-Na/K phosphate,
pH 7.2). A column (3 × 30 cm) was prepared and calibrated with bovine serum albumin
(tool. wt. 67ooo) and ribonuclease (mol. wt. I3 5oo). Ioo units of interferon were chromatographed in the presence of the marker proteins (Fig. r a). The apparent mol. wt. of the most
active fractions were 72 ooo and 26 ooo. Chromatography of 2oo units of interferon in
the absence of the marker proteins revealed one region with a mean mol. wt. of 25 ooo (Fig.
I b). Other interferons are known to be able to bind to bovine serum albumin (Huang et al.
I975) and it appears likely that the material eluting with an apparent mol. wt. of 76o0o is
a complex of interferon bound to bovine serum albumin.
Taber, Alexander & Whitford (I976) derived a line of Chinese hamster ovary cells
(CHO-KI : CCI-6I) which was persistently infected with a reovirus, and was resistant to
superinfection by a number of unrelated viruses. Several investigators have suggested a
major role for interferon in the maintenance of persistent virus infections (Walker, I964;
Thacore & Youngner, I969) and it seemed of interest to establish whether CHO-KI cells
possess an interferon system. No interferon was detected after treatment with the following
viruses: Sindbis, Semliki Forest, vaccinia (all tested at so and 200 p.f.u.]cell); mouse
encephalomyocarditis and Mengo (both at I and Io p.f.u./cell) and u.v.-NDV (2oo to Iooo
particles/cell). Ageing in vitro had no effect and poly (rI).poly (rC) (o-• to Ioo/zg]ml) in
the presence and absence of DEAE dextran (Io and Ioo #g]ml) did not induce resistance to
challenge with Sindbis virus. Thus CHO-KI cells appeared to be unable to synthesize
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353
Table I. Interferon production and action
Protection against Sindbis virus
(dye uptake as a % of uninfected
cell controls) after treatment with
Interferon (OUs0/IOs cells) produced
after treatment with
~r
Poly (rI). Poly (rC)]"
u.v.-NDV*
150
o
11
o
4
o
0
2
0
I
0
5
0
o
Cells
Embryo
Lung
Kidney
Heart
Iris
Skin
CHO-Kt
Human fibroblasts
WI-38
A
r
Chinese hamster
interferon$
94
75
---
Poly (rI). Poly (rC)t
50
50
42
IO
IO
IO
o
-
-
-
-
92
--
--
0
--
--
0
200
to
IOOO
particles/cell.
t ~oo/zg/ml.
$ 75 DUso units/culture.
!/
0
20
40
6O
80
I
I
100
0
I
(a)
3
o
20
40
J
I
60
80
100
[
I
----]
(h]
1
i
%
1-
40
30 --
I
20 .:
"v-
10
Elution vol. (ml)
Fig. I. Gel filtration chromatography of interferon on Sephadex G-I5O in the presence (a) and
in the absence (b) of bovine serum albumin and ribonuclease. - - , E2so, O - - O , interferon
activity. Optical density peaks: fraction 24, bovine serum albumin marker (67o00); fraction
04, ribonuclease marker (I 3 5oo).
interferon. They did, however, respond to both Chinese hamster interferon a n d h u m a n
leukocyte interferon. I n this regard they resemble the V E R O line of African G r e e n M o n k e y
cells (Desmyter, Melnick & Rawls, I968). Preliminary studies (M. J. M o r g a n , u n p u b l i s h e d
data) suggests that C H O - K I cells c o n t a i n the structural gene for interferon synthesis, albeit
i n a n inactive state. Clones have been obtained of hybrids of C H O - K I cells and h u m a n cells,
a n d some of these clones are able to synthesize Chinese hamster interferon. Thus, the gene
for interferon synthesis could be activated by a h u m a n c h r o m o s o m a l product. K a o & Puck
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(1972) described an analogous event in hybrids of Chinese hamster and human cells where
the presence of human chromosome 4 or 5 allowed the synthesis of a hamster esterase not
found in the parental cells. Fmther results and details of the isolation and characteristics
of these hybrids are to be published later.
The role of interferon in the lzersistent infection of CHO-KI cells with a reovirus (Taber
et al. 1976 ) remains unclear, but since hybrid cells can synthesize hamster interferon, it is
possible that some is made by the persistently infected cells.
This investigation was suppolted by a grant from the Medical Research Council. Part of
the work was performed at the Eleanor Roosevelt Institute for Cancer Research, Denver,
U.S.A., during the tenure of an Eulopean Molecular Biology Organisation Short-term
Fellowship. I thank Dr T. T. Puck for his hospitality, Mrs Lynne Stubbs for excellent
technical assistance, and Flow Laboratories for a gift of medium and Petri dishes.
Department o f Biochemistry,
University o f Leicester,
Leicester L E I 7RH,
England
M . J. MORGAN
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