589
J. gen. ViroL (I979), 42, 589-595
Printed in Great Britain
Effects o f Leukocyte and Fibroblast Interferon on Events
in the Fibroblast Cell Cycle
By E. L U N D G R E N ,
I. L A R S S O N , H. M I O R N E R
AND ~). S T R A N N E G A R D
Institute of Pathology and Department of Virology,
University of Umed, Sweden
(Accepted 19 September I978)
SUMMARY
Serum-depleted human foetal skin fibroblasts were stimulated by addition of
IO ~ foetal calf serum to proliferate synchronously for at least one cell cycle. This
proliferation was suppressed by leukocyte or fibroblast interferon (IF), which
prolonged the GI phase and diminished the rate of D N A synthesis during the
S phase in a dose-dependent manner. When used in identical concentration, as
judged in terms of units of antiviral activity, fibroblast IF had more pronounced
effects on cell cycle events than leukocyte IF. Interferon exerted its effect in early
GI, before the cells were irreversibly committed to DNA synthesis.
INTRODUCTION
Interferons (IF) have been shown to inhibit the growth of proliferating cells, the so-called
anticellular activity (Paucker et al. 1962; Gresser et al. I97oa). This activity of IF has
mostly been studied in transformed cell lines (Gresser et al. I97oa, b; Adams et al. 1975) but
has also been found with normal cell lines and even with primary cultures (Lindahl, I974;
Dahl & Degr6, 1976). Such non-neoplastic cell lines are subject to a strict growth control,
and some important details of the mechanisms involved have been worked out (Smith &
Martin, 1973 ; Pardee, 1974; Lindgren et al. 1975). In attempting to define the mechanism of
the anticellular action of IF, we have therefore chosen to use proliferating normal cells. In
the present paper we report that IF prolongs the GI phase and reduces the height of the
S peak by reducing the number of cells in the S phase. The point of attack of IF seems to be
some early events during GI.
METHODS
Interferon. Partially purified human leukocyte IF (PIF) with asp. act. of 2"5 × lO5 units/mg
protein was kindly supplied by the Finnish Red Cross Blood Service. Fibroblast IF was produced by inoculating human embryonic fibroblast cell cultures with 6oo haemagglutination
units per ml of Sendai virus. The medium from inoculated cultures was harvested after 24 h
and then dialysed, first against o. I M-glycine-HC1 buffer, pH 2.0, for 24 h and then against
phosphate-buffered saline, p H 7"3, for 48 h. The preparation was concentrated by precipitation with 3o ~ polyethylene glycol 40o0 to achieve a sp. act. of 2. 5 × Io 4 units/mg protein.
Interferons were assayed on amnion U cells in microtrays with vesicular stomatitis virus
as described by Havell & Vil~ek (1972). An internal laboratory reference IF standard,
calibrated against the research standard 69/I9 (National Institute for Medical Research,
London, England), was included in each assay.
oo22-I317/79/oooo-2888 $02.00 © 1979 SGM
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590
E. LUNDGREN AND OTHERS
Cell and culture conditions. The fibroblast cell lines were derived from the skin of human
foetuses from abortions induced during the 16th to 24th weeks of pregnancy. The cells were
used between the 5th and 2oth passage. Cell lines from five different foetuses were used, but
no differences between them were found in the parameters measured.
Cells were grown in 90 mm plastic dishes (Nunc, Roskilde, Denmark) with Eagle's
minimum essential medium (MEM) supplemented with antibiotics and Io % foetal calf
serum (FCS), unless otherwise stated. The cultures were incubated in humidified air with
5 % CO2 at 37 °C. The cells were split in a I :2 ratio every 3rd or 4th day.
Assay of cell proliferation
Thymidine incorporation. The fibroblast cells were suspended in MEM containing IO %
FCS and seeded at a density of 5 × IO cells/cm 2 growth surface in microtrays (Falcon
Plastics Co., Oxnard, California). Three days later, when the cell number was about
I'5 × Io 5 cells/cm 2, the medium was changed to MEM with o'5 % FCS and the cultures were
further incubated for 24 h (the cells at this stage are termed ' serum-deprived' cells in this
paper). The experiments started with a change to medium with IO % FCS (serum stimulation) or as described in the Results. At the appropriate time later the cells in the microtrays
were pulsed for 6o rain with I/~Ci/ml tritiated thymidine (3H-dThd; sp. act. 6I mCi/mM)in
MEM without serum. The cultures were then trypsinized, washed and precipitated by
TCA to a glass fibre filter by a semi-automatic multiple sample processor (Hartzman
et al. I972).
Autoradiography. Cells were grown on coverslips placed in Linbro tissue culture trays
(FB-24TC, Flow Laboratories, lrvine, Scotland) in MEM with Io % FCS, with or without
IF, and containing I #Ci/ml of 3H-dThd. After 24 h the cultures were fixed in methanolacetic acid for I h, washed in 7o % ethanol, air dried and dipped in Ilford G5 emulsion
(Ilford Ltd, Ilford, England). After an exposure time of 2 to 3 weeks the autoradiograms
were developed, stained in May-Griinewald:Giemsa and counted in duplicate by light
microscopy using an oil-immersion objective. Only cells with more than Io to 20 grains over
the nuclei were counted as S phase cells.
Cell number estimation. Cells were seeded at a density of 1.5 x to 5 cells/cm 2 growth
surface. After different time periods in the experimental media, the cells were trypsinized
and counted in an electronic cell counter (Linson Instrument AB, Stockholm, Sweden).
RESULTS
Effects of serum on cell growth
When cells serum-deprived for 2 4 h were incubated for a further 2 4 h with fresh medium
containing different amounts of serum, the rate of 3H-dThd incorporation increased
progressively with the serum concentration and reached a maximum with Io % FCS. In a
similar experiment, cell numbers were determined after 7 2 h of growth with different serum
concentrations. A threefold increase in cell number was achieved with Io % FCS (Fig. I).
There was at least one additional round of cell division in all the cultures if they were then
re-fed with Io % FCS, regardless of the previous serum concentration. Therefore, with
Io % FCS the rate of D N A synthesis and the increase in cell number was optimal; also, the
cells were not adversely affected by exposure to low serum concentration. Therefore, in the
following experiments, growth arrest and growth stimulation was achieved by culturing the
cells in o'5 % and Io % FCS, respectively.
Fig. 2 shows the kinetics of SH-dThd incorporation after shift from o. 5 % to IO % FCS.
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Interferon and cell cycle events
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Fig. I
Fig. z
Fig. I. Effect of foetal calf serum (FCS) on cell number (calculated as the cell density in the Petri
dish). Serum-deprived cells were re-fed with fresh medium containing FCS in the indicated concentrations. ©--©, Cell number after 72 h; 0 - 0 , cell number after another 7z h period, during
which the serum concentration in the medium in all the cultures was changed from that indicated to
Io % FCS.
Fig. z. Kinetics of 3H-dThd incorporation in serum-deprived cells cultured in IO % FCS. At the
times indicated after change to ~o % FCS the cultures were pulsed with ZH-dThd.
The pre-replicative G1 phase as measured from the start of serum stimulation to the 50 %
point of the ascending limb of the 3H-dThd incorporation curve amounted to I9"3 _+o'4 h.
The S phase, measured as the distance between the 5o % points on the ascending and
descending parts of the curve, lasted i4.8_ I.O h. In many experiments a second wave of
synchronous DNA-synthesis was easily discernible.
Effect of IF on cell growth
Fig. 3. shows that IF preparations had a marked effect on cell growth. When IOOO
reference units/ml were used, leukocyte IF reduced the increase in cell number during
I44 h by 34 %. An equivalent amount (in terms of antiviral units, as measured on amnion U
cells) of fibroblast IF had a bigger effect: the increase in cell number was reduced by 9o %.
These effects were abolished by treatment of the IF preparations with trypsin, but not by
treatment with RNase and pH 2.
To analyse this effect on growth, the rate of ~H-dThd incorporation into TCA-precipitable
material was studied during the cell cycle. Fig. 4 shows that when leukocyte IF was added to
serum stimulated cultures, G I was prolonged and the height of the S peak was depressed in a
dose.dependent way. There was no obvious change in the duration of the S phase. Fibroblast IF had a similar dose-dependent effect on G1 and S phases (data not shown).
Table I shows that even when serum and IF had been removed, I F still affected uptake of
"H-dThd into the cells during a 6o min pulsing period. At a concentration of IOOOunits/ml
both interferons reduced the TCA-soluble 3H-dThd pool by 75 %. However, the pool size
was the same after Io h in Io % FCS as after 24 h, whether or not the cells displayed an
S peak during the different treatments. Thus, transport of thymidine into the cells did not
seem to be the rate limiting step. Similar results were obtained after 13 and 17 h (data not
shown).
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592
E. L U N D G R E N
AND
OTHERS
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Time after serum stimulation (h)
Fig. 3
Fig. 4
Fig. 3. Effect of interferon on serum-stimulated cell growth. Serum-deprived cells were re-fed at the
start of the experiment with fresh medium containing IO ~ FCS and IOOOunits/ml leukocyte IF
(ll--II), or moo units/ml fibroblast IF ( A - - A ) , or no IF ( O - - © ) . Cell numbers are calculated as
the cell density in the Petri dish.
Fig. 4. Effect of leukocyte IF on cell cycle kinetics. Experimental conditions as in Fig. 3. Cells were
cultured in absence of IF ( © - - © ) , or in presence of 50 units/ml (O--O), 25o units/ml (ll--iI),
500 units/ml ( A - - A ) , or IOOOunits/ml ( T - - T ) of IF.
T a b l e I.
Uptake of 3H-thymidine by control and interferon-treated cells*
Uptake of nH-dThd
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Treatment
Control]
Leukocyte IF
Fibroblast IF
Duration
(h)
into
TCA-precipitable
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into
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244 + r I
282+ 36
* Fibroblast cells were grown in IO Yo FCS in the presence of the indicated interferon at tooo units/ml for
IO h or 24 h and were then pulsed with aH-dThd for 60 min in medium containing no serum or interferon.
The mean ct/min ( + s.e.) from four replicate cultures are shown.
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Interferon and cell cycle events
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Time after serum stinmlation (h)
Fig. 5
Fig. 6
Fig. 5. Effect of IF on the entry of cells into S. Serum-deprived cells were stimulated with medium
containing IO ~ FCS in the presence of aH-dThd (t #Ci/ml). At the times shown the cells were
fixed and autoradiograms were prepared. O - - O , control; IlI--L Iooo units/ml leukocyte IF;
& - - A , Iooo units/ml fibroblast IF.
Fig. 6. Comparison between the effects of serum depletion ( O - - O ) and interferon addition (O---I)
on cellular D N A synthesis. At the indicated times cultures were changed to serum-free medium, or
leukocyte interferon (25oo units/ml) was added. Incorporation of 3H-dThd was determined 24 h
after time o in all cultures by pulsing for I h.
Effects of IF on rate of entry into S phase
Autoradiography experiments were performed with cells labelled with aH-dThd for 24 h
after stimulation with Io ~ FCS in the presence or absence of lOOOunits/ml of leukocyte or
fibroblast IF. The results are represented in Fig. 5 according to the suggestions of Smith &
Martin (I973) in terms of the fraction of unlabelled cells after different time periods, i.e. the
fraction of cells remaining in G1 or the A state.
During the first 16 h there was a small decrease in the proportion of cells in GI (unlabelled
nuclei). Then the slope changed, indicating that the cells started to enter S at a constant rate,
calculated as o'o45 h -1. With cells treated with Iooo units/ml leukocyte IF, there was a
delay before the cells changed their rate of entrance into S (constant estimated as o-oi6 h-0.
In contrast, during the observation period of 3o h, all the cells treated with Iooo units/mI
fibroblast IF remained in G1. At comparable time periods, there was no obvious difference
in the number of grains per nucleus, which indicates that IF did not exert its effect mainly by
decreasing phosphorylation of thymidine. Therefore, our method of estimating 3H-dThd in
TCA-precipitable material seems to be a reasonable way to estimate the rate of D N A
synthesis.
The G1 phase of the cell cycle can be subdivided into two distinct stages. This is illustrated
by the experiment in Fig. 6, in which IO ~ FCS medium was added to all cultures at the
start. At the indicated times the serum-containing medium was changed to medium devoid of
FCS. aH-dThd incorporation was estimated by pulsing all cells after 14 h. After 11 h, but
not after 7 h, of continuous serum stimulation, there was a significant increase in D N A
synthesis. Calculated from the 5o ~ point of the ascending limb, it appears that the cells
were able to pass the G I / S transition point after 14"5 + o.6 h of continuous serum stimula-
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594
E. L U N D G R E N
AND OTHERS
tion. As G~ was I9"3 h, the serum-independent period, corresponding to the Glc period of
Temin (I97I), was 4"8 h. The same experiment was performed but with the addition of
leukocyte IF at various times instead of a change to serum-free medium. As demonstrated in
Fig. 6, addition of IF had the same effect on cell proliferation as serum depletion. Thus, IF
suppressed DNA synthesis only if added before the start of the Glc phase.
DISCUSSION
Few and seemingly conflicting data are currently available concerning the effects of IF
preparations on cell cycle events. From studies of the effect of IF on Li2ro cells, MacieiraCoelho et al. (i97 I) suggested that IF delays the entry of cells into the division cycle.
Killander et al. (I976), however, reported that LI2iO cells were arrested in all stages of the
cell cycle and not in a particular stage, and that the proportion of cells in the different stages
were the same as in control cultures. However, these authors did not comment on their
finding that it was more than 24 h before the cell number was decreased; this could mean
that IF had to await specific cell cycle events to express its effect. Fuse & Kuwata 0976)
claimed that virus-transformed human fibroblasts were blocked in the transition from G t to
S if leukocyte IF was given late in GI. In L cells synchronized by double thymidine block,
IF delayed the S phase by several hours if given when the block was released, i.e. at the
G I / S transition (O'Shaugnessy et al. I972).
In these cited studies, only neoplastic cell lines were used and these may have defects in
the control of the cell cycle. We have used serum-stimulated and non-neoplastic fibroblast
cultures, which are subject to strict growth control, and our results are apparently different,
for reasons which are not yet clear.
Several authors have pointed out the presence of two distinct restriction points in cell
lines with strict growth control (Smith & Martin I973; Pardee, I974; Lindgren et al., I975).
One point is at the transition from the resting stage into the cell cycle (Go/GI), a step
normally initiated by several hormone-like growth stimulating factors and serum. When the
cells have passed the other point (transition from GI proper to GIc), they start DNA
synthesis irreversibly and independent of the presence of these stimulatory factors.
Our results indicate that the primary point of attack of fibroblast and leukocyte IF in
diploid, non-neoplastic fibroblasts is during the pre-replicative GI phase, prior to GIc.
Once the cells have passed the GI proper/Gic transition, they do not seem to be sensitive to
the anticellular effect of IF.
IF exerted its effect during the growth factor dependent part of GI, but had no effect on
DNA synthesis if added during GIc. It therefore seems certain that the prime target of
attack is on the triggering system regulating cell proliferation and not on the process of DNA
synthesis. Several interesting possibilities can be suggested to explain this: IF may compete
with growth factors for receptors on the cell surface; it may diminish the number of receptors
for growth factors; or it may alter the affinity of the receptors for growth factors. Our
experimental data give no answers to these questions, but hint that interferon may be an
important probe for the mechanism of growth control in normal and neoplastic tissue.
During the revision of this manuscript a report by Sokawa et aL (I977) showed similar
interferon effects on the cycle of 3T3 cells, but in their system the GI phase was not
prolonged.
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Interferon and cell cycle events
495
The study was supported by a grant from AB Kabi, Stockholm, Sweden, and Lion's
Research Foundation Dept. of Oncology, University of Umeh, Sweden. We thank Miss
Christine Roth and Miss Inger Eriksson for skilful technical assistance.
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rUSE, A. & KUWATA,T. (I 976). Effects of interferon on the human cell line, RSa: inhibition of macromolecular
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(Received 20 March 1978 )
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