Experimental Cell Research 159 (1985) 27-36

Expression of the 2-5A System during the Cell Cycle

VALERIE WELLS and LIVIO MALLUCCI Department of Microbiology, Guy's Hospital Medical School, London SE1 9RT, UK

To determine whether the 2-5A system has a role in the regulation of cell growth we have examined all constituents of the 2-5A pathway in mouse embryo fibroblasts undergoing one cycle of division at the tertiary stage under conditions where a high degree of uniformity is maintained within each stage of the cycle. Levels of the 2-5A synthetase increased up to tenfold late in S phase and declined as cells moved through G2. A similar but smaller increase in the 2-5A-dependent ribonuclease was observed, whereas activity of the 2'5' phosphodiesterase was highest in quiescent cells. At the time of maximum synthetase levels no phosphorylated 2-5A could be detected in the intact cell. Endogenous interferon (IFN) was found in the culture supernatants in increasing concentration with cell cycle progression and addition of antibodies to IFN reduced the increase in synthetase seen in late S, Treatment of cells with a growth inhibitor that cells produce also affected synthe- tase activity. © 1985 Academic Press, Inc.

The problem of how the growth of cells is controlled remains largely unre- solved although many changes which occur as cells progress through the cell cycle are well documented. One biochemical system which has been proposed to have a role in the growth-inhibitory action of interferon is the 2-5A pathway. This consists of a 2-5A synthetase which polymerizes ATP into a series of 2'5'-linked oligomers of the series ppp(A2'p),,A(2-5A), 2-5A per se which specifically acti- vates a 2-5A-dependent ribonuclease (RNase) to cleave both ribosomal and messenger RNA (rRNA and mRNA) and a 2'5' phosphodiesterase which de- grades 2-5A ([1-3] for reviews). (2-5A is used here to cover all components of the series px(A2'p),A for x=2-3 and n~>2. The non-phosphorylated components (x=0) will generally be referred to as 'core'.) Treatment of cells with interferon results in a substantial increase (10 to 10 000-fold) in levels of the 2-5A synthetase [4, 5]. In JLS-V9R cells a significant increase in the RNase has also been reported [6]. In the absence of interferon treatment studies of cells under different condi- tions of growth have shown that 2-5A synthetase activity is greater in confluent and in serum-starved cells than in subconfluent cells or in cells stimulated by serum [7-9]. Also in JLS-V9R cells levels of the 2-5A-dependent RNase are higher when cells have reached confluence [10]. On the other hand, studies on human diploid fibroblasts have shown that stimulation of growth by epidermal growth factor (EGF) increases synthetase levels [11]. Exogenous non-phosphory- lated 2-5A (core) has been reported to inhibit the mitogenic response of mouse lymphocytes [12] and BALB/c 3T3 cells [13], although there is a report where its action appears not to involve the 2-5A-dependent ribonuclease [14]. Other at-

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

0014-4827/85 $03.00

28 Wells and Mallucci

t e m p t s to d e t e c t an e f fec t o f c o r e on the m i t o g e n i c r e s p o n s e to s e r u m and g rowth

fac to r s in Swiss 3T3 ce l l s have no t m e t wi th s u c c e s s [15]. N e v e r t h e l e s s , t h e r e

r e m a i n s c o n s i d e r a b l e i n t e r e s t in w h e t h e r the 2-5A s y s t e m has a ro le in the

r egu la t i on o f cel l g rowth .

A use fu l a p p r o a c h to t he p r o b l e m w o u l d be to e x a m i n e all c o n s t i t u e n t s o f the 2-

5 A p a t h w a y in a s i t ua t i on w h e r e s equen t i a l c h a n g e s in g rowth s t a t e can be

fo l lowed u n d e r c o n t r o l l e d c o n d i t i o n s . W e have r e c e n t l y d e v e l o p e d a cel l c y c l e

s y s t e m w h e r e , b y v a r y i n g cu l t u r e c o n d i t i o n s r a t h e r than us ing d rugs o r s e r u m

s t a rva t i on , a h igh d e g r e e o f p o p u l a t i o n u n i f o r m i t y is m a i n t a i n e d wi th in e a c h s t age

o f the c y c l e in m o u s e e m b r y o f i b r o b l a s t s s y n c h r o n o u s l y p r o g r e s s i n g t h r o u g h one

c y c l e o f d iv i s ion at the t e r t i a r y s t age [16-18]. In add i t i on , wi th in th is s y s t e m cel l

g rowth can be i n h i b i t e d b y a g r o w t h - i n h i b i t o r y p r o t e i n tha t we have r e c e n t l y

i s o l a t e d and tha t is p r o d u c e d b y the cel ls p o s s i b l y as a nega t ive g rowth m o d u l a t o r

[19]. U s i n g this s y s t e m we have e x a m i n e d w h e t h e r a c o r r e l a t i o n ex i s t s b e t w e e n

m o v e m e n t o f ce l l s t h r o u g h the c y c l e and levels o f c o n s t i t u e n t s o f the 2 -5A

p a t h w a y and have c o n s i d e r e d the r e l a t i o n s h i p wi th e n d o g e n o u s i n t e r f e r o n and

the i n v o l v e m e n t o f the g r o w t h inh ib i to r .

M A T E R I A L S A N D M E T H O D S

Cell Cultures

Cultures of embryo fibroblasts were prepared from mice of the C57 B16 strain. Synchrony was obtained avoiding the use of metabolic inhibitors, controlling cell growth by varying the cultural conditions throughout the primary, secondary and tertiary cultivations. Details of the procedures have been reported previously [16-19]. Briefly, taking into account the efficiency of cell plating, cells were seeded at a density such that when those capable of dividing had undergone one division cycle a confluent monolayer was obtained. Primary cells were seeded in Eagle's BHK medium containing 10% tryptose phosphate broth and 10% newborn calf serum (growth medium) in an atmosphere of 5 % CO2 in air. Cell division occurred at bout 30 h and the cultures were left for a further 24 h. These were subcultured and after cell division had occurred (at about 28-30 h) the growth medium was changed to medium containing 2 % newborn calf serum and cultures were maintained in this condition for a further 48 h to provide confluent quiescent G1 populations as assessed by cytofluorimetric analysis and autoradiography. These cells were subcultured and seeded at half the density expected at confluence in tissue culture flasks or in scintillation vials in medium containing 5 % fetal calf serum (FCS) and were incubated in a water bath at 37°C to undergo one cycle of division.

Thymidine Incorporation

Cultures in scintillation vials were incubated in a 37°C water bath and were pulse-labelled for 30 min with 0.2 ~tm [3H]thymidine ([methyl-3H]thymidine, 18-25 Ci/mmol, Radiochemical Centre, Amer- sham, Bucks, England). Uptake into acid-precipitable material was assessed as described previously [16] and related to cell number.

Cellular DNA Content and Population Distribution

Cellular DNA content was quantitated by cytofluorimetry [20] and the percentage of cells in each stage of the cycle was assessed at intervals throughout one division cycle. Cells were removed from the glass using a 0.02 % solution of ethylenediaminetetra acetic acid (EDTA) containing 0.1% trypsin. After neutralization with serum the cells were centrifuged and washed twice with phosphate-buffered saline (PBS) at 4°C, fixed in 70% ethanol in PBS and then resuspended in 20 ~tg m1-1 mithramycin in 25 % ethanol containing 15 mM MgC12 for cytofluorimetric analysis as employed in previous studies [17, 181.

Exp Cell Res 159 (1985)

Cell cycle expression o f the 2-5A system 29

Cell Number Cell number was assessed in cultures removed from the glass using 0.5 ml 0.02 % EDTA, containing

0.1% trypsin plus 0.2 % trypan blue, and neutralized with serum. Unstained live cells were counted in a Fuchs-Rosenthal haemocytometer. In all cases stained cells were less than 2 % of the total cell number.

Preparation o f Growth Inhibitor The mouse embryo fibroblasts produce a growth inhibitor which reversibly inhibits cell growth and

which exhibits cell type specificity [ 19]. The inhibitor has the character of a heat-labile non-dialysable protein, requires protein synthesis for its production and is shed to the medium. A semi-purified fraction containing two major proteins of molecular weight (MW) 12 500 and 15 000 was separated from serum-free conditioned medium obtained from confluent quiescent secondary GI cultures. Details of the isolation procedures and the method of assessment of inhibitory activity have been described recently [19].

Enzyme Assays For enzyme assays cells were scraped from the glass substrate, washed twice in serum-free

medium, centrifuged at 10 000 g and the cell pellets frozen and stored at -70°C. The pellets were then thawed with gentle vortexing into approx. 1.5 vol of buffer containing 50 mM KC1, 10 mM HEPES, pH 7.6, 1.5 mM Mg 2+, 7 mM mercaptoethanol and 0.5 % Nonidet P40, centrifuged at 10 000 g for 10 min and the supernatants harvested. For pellets from 107 cells 70 ~1 of buffer was used, and in each experiment the cell extracts to be compared were adjusted to the same absorbance at 260 nm.

2-5A Synthetase

Twenty ktg of the cell extracts were incubated for 1 h at room temperature with an equal volume of poly I poly C cellulose suspension in a buffer containing 50 mM KC1, 10 mM HEPES, pH 7.6, 1.5 mM Mg 2+ and 20 % glycerol. Following binding of the enzyme the poly I poly C cellulose was washed three times with the same buffer and then incubated overnight at 30°C in the same buffer containing 7 mM mercaptoethanol and 3 mM ATP. The 2-5A produced was then assayed using a radiobinding (RB) assay developed by Kerr et al. [21].

2-5A Binding Protein (2-5A-Dependent Ribonuclease) Ten ~tl of the cell extracts were incubated with 3000 cpm of ppp(A2'p)3A3 [32p] pCp (1-3× 106

Ci/mol) for 1-2 h at 4°C and the 32p bound to the protein was measured by its retention on nitro- cellulose filters [21].

2'5' Phosphodiesterase

The degradation of 2-5A by the enzyme was assayed as described for extracts of Ehrlich ascites tumour cells [22] by incubating cell extracts with ppp(A2'p)nA for times from 20 to 180 min at 30°C. The incubation conditions included 1 mM ATP to protect the 5' triphosphates of 2-5A. At the end of each incubation period samples were heated to 90°C for 5 min and then centrifuged at 10000 for 5 min. The remaining ppp(A2'p)nA was monitored using the radiobinding assay [21].

Assay of 2-5A

For the assay of 2-5A, frozen pellets of cells prepared as described above were thawed into approx. 2 vol. of 7.5% TCA and diluted further with 5% TCA. After centrifuging, the supernatants were neutralized with octylamine : freon and extracted with freon. The neutralized aqueous samples were then freeze-dried and taken up in 10 mM HEPES buffer, pH 7.0 to be assessed by the various assays for detection of the components of the oligomeric series of 2-5A.

Interferon Assay

Dilutions of media from tertiary fibroblasts progressing through the cycle were added to confluent monolayers of L cells in 16 mm diameter multiwell plates. After 16 h incubation the media were

Exp Cell Res 159 (1985)

30 Wells and Mallucci

removed and the cells infected with encephalomyocarditis virus (EMC) at a multiplicity of 0.5 pfu per cell in medium containing 0.5% fetal bovine serum and 5 Ixg ml -~ actinomycin D, a dose which reduced cellular incorporation of [3H]uridine to background levels within 1 h. After 3 h the cultures were pulsed with [5-3H]uridine (0.2 ~tM, 26 Ci/mmol) for 2 h, a suitable period as assessed from kinetics of viral RNA synthesis. The medium was then removed, the monolayers washed twice with cold PBS, extracted with 5 % TCA at 4°C and washed with water. They were then solubilized in 0.1 M NaOH and aliquots in scintillation fluid assessed for radioactivity. The optical density of the remainder of the sample was read at 280 nm in order to adjust readings to the same protein concentration.

The antibodies to mouse interferon were a gift from Dr I. Gresser. They consisted of a purified IgG fraction of antibodies to fibroblast interferon pre-adsorbed to mouse cells and had a titre of 1.6× 10 -5 against 4 units of interferon [23].

RESULTS

The starting point of the cell system used in these studies were Gl-quiescent secondary fibroblasts obtained following controlled growth during primary and secondary cultivations (see Materials and Methods). When subcultured these cells gave a population of tertiary fibroblasts which moved through one cycle of division with a high degree of synchrony as assessed by cytofluorimetric analysis, incorporation of [3H]thymidine and cell number (fig. 1). Cells entered S phase at 12 h, advanced through S phase to enter G2 at about 22 h, while cell number increased between 25 and 30 h to remain at a constant level. The effect of the growth inhibitor on these cells is shown in table 1.

Enzymes of the 2-5A System

Several experiments were carried out and each gave a similar profile of enzyme activities. The results obtained in one typical experiment are presented in figs 2 and 3.

2-5A synthetase. The activity of this enzyme at various stages of the cell cycle was assessed in extracts of stationary G1 cells and of cells progressing through the cycle by quantitating 2-5A synthesized from ATP using a radiobinding assay [21]. Fig. 2a shows that activity was low in cells stationed in G1, in cells progressing through G1 and during the earlier part of S phase after which the activity increased up to tenfold at the peak of S phase and remained high during

Table 1. Effect of growth-inhibitory factor a on cell cycle progression

Time of addition of factor (40 ~tg ml -~) Percentage reduction of cells in S and G2 at (hours after seeding) 12 h 18 h 21 h 24 h 29 h

Percentage reduction of growth at 40 h

4 0 - 4 3 + 1 . 3 7 - -

1 2 - 14+2.1 - 65.5+1.04 - 18 . . . . 52.5+1.25

43+1.32 63+1.09 71+1.02

a For information on heat lability, reversibility of action and specificity, see [19].

Exp Cell Res 159 (1985)

Cell cycle expression of the 2-5A system 31

o

8

6

4

2 ×

80

~ 60

,E 40

u

~0

20

a

" - I I . . . . . . , , , I ~ 1 ,

b

- - / I . . . . . . . . - r / / - ~ - / I , 12 15 18 21 2~ 27 30 33 36 48 72

Time af ter Seeding ( h o u r s )

.80 25

o° ~ -~0 ~ ~ 20

< 5'

-40 C~ <~ 15

-5 20 ~ 10

5

o"

= ~ "2.0 a.

m

.0 T~

S

×

E

~ ' 1 1 . . . . . . . . . I I , I I d

- - 4 1 . . . . . . . . . I I , II , 12 15 18 21 24 27 30 33 36 48 72

Time af ter Seeding ( hou rs )

Fig. 1. (a) Incorporation of [3H]thymidine into acid-insoluble fraction in II, GI quiescent secondary cells; 0 , throughout the cell cycle at the tertiary stage; [3, percentage of cells in G1 in confluent secondary cells; ©, in tertiary cells during the cell cycle calculated from cytofluorimetric analysis. (b) Percentage of cells in S+G2 in II, confluent secondary cells; @, throughout the cell cycle at the tertiary stage calculated from cytofluorimetric analysis, and O, cell number during the cell cycle. Fig. 2. (a) Activity of 2-5A synthetase in @, GI quiescent secondary cells; ©, throughout the cell cycle at the tertiary stage expressed as nmol 2-5A synthesized from ATP over a period of 20 h. (b) Levels of 2-5A-binding protein (RNase) in II, G1 quiescent secondary cells; [3, throughout the cell cycle at the tertiary stage, expressed as the amount of ppp(A2'p)sAY [32p]pCp bound by the cell extracts.

the rest of S. Levels then decreased sharply as cells progressed through G2 to remain at a constant level as cells returned to and stayed in GI.

The 2-5A-binding protein (RNase). As the ability of the 2-5A-binding protein to bind 2-5A co-purifies with the 2-5A-dependent RNase throughout several purification steps [24] it is generally accepted that estimation of the bind- ing protein reflects levels of the RNase. For assessment of the RNase cell extracts were therefore incubated with a radiolabelled derivative of 2-5A, ppp(A2'p)3A3'[32p]pCp, to determine their binding capacity. Similar levels of the 2-5A-binding protein were observed in G1 cells, whether arrested in G1 or moving through this stage of the cycle. Levels then rose after the cells had entered S phase to a twofold higher value falling off slightly thereafter (fig. 2 b).

The 2'5' phosphodiesterase. The 2'5' phosphodiesterase degrades 2-5A. Its activity was assessed by adding 2-5A to cell extracts and monitoring the kinetics of its degradation by assaying the remaining 2-5A by the radiobinding assay. Fig. 3 shows that enzyme activity was higher in confluent quiescent G1 cells than in cells undergoing progression through the cycle.

3-858337 Exp Cell Res 159 (1985)

32 Wells and Mallucci

~ V 0 • mO ~ V

I \ No %*... ~]-~ I \ % - -~oj-

"~ 60 - __ T 0 •

, , . . . . I I ~ - / t - - - - 7 3

10 20 30 ~0 50 60 120 180

Time (m inu tes )

5

~o

3 0 -

2 0

1 0 . t

-----4/----,- 12

, i i , 1 7 15 18 21 2q 2

Time of Ha rves t of C u l t u r e Medium (Hou rs a f te r Seeding)

Fig. 3. Kinetics of degradation of 2-5A by 2'-5' phosphodiesterase. Extracts from A, confluent G1 secondary cells; confluent G1 tertiary cells after O, 48; O, 72 h of culture; A, V, [3, II, O , ~ , tertiary cells harvested at 12, 15, 18, 22, 25, 30 and 36 h after seeding. Fig. 4. Assessment of activity to reduce EMC virus RNA synthesis in medium from 0 , G1 quiescent secondary cells; ©, cells progressing through the cell cycle.

Levels o f 2-5A

As levels of the enzymes of the 2-5A pathway varied, with the 2-5A-dependent RNase increasing where higher synthetase activity was observed, levels of 2-5A per se were also assessed.

Cell extracts were assayed for the different components of ppp(A2'p)nA by a radiobinding (RB) method which detects predominantly (p)pp(A2'p),A (where n=2--4) [21]. Levels of what appeared to be approx. 3 nM in trimer equivalents were detected in extracts from 10 7 cells. As values of less than 20 nM in crude extracts can be misleading (reflecting the presence of non-specific interference) large scale preparations (108 cells) were made at selected times. Cells stationed in G1 and cells at late S stages were used where differences in the synthetase and nuclease levels were most marked. Low positive signals in the RB assay were again obtained. However, the presence in the extracts of an inhibitor of 2-5A- dependent rRNA cleavage prevented assessment of functional 2-5A by a RNA cleavage assay [25]. For further analysis the extracts were fractionated by high performance liquid chromatography (HPLC) and assayed by the RB method, by a radio-immunoassay which shows maximum sensitivity to any compound which contains the moiety p(A2'p), A [26] and by the rRNA cleavage assay for function- al 2-5A ((p)pp(A2'p),A (n=2--4) [25]. Negative results were obtained throughout. On the basis of these the presence of 5'mono-, di- or tri-phosphorylated compo- nents of 2-5A can be excluded down to nanomolar levels (i.e., 1 pmol g wet weight-1) in the intact cells.

The HPLC fractions were also assessed for the presence of non-phosphorylat- ed 2-5A using a radio-immunoassay which detects (A2'p),A (n>~2) with highest

Exp Cell Res t59 (1985)

Cell cycle expression of the 2-5A system 33

Table 2. Effect of treatment of cells with antibodies to IFN on levels of 2-5A synthetase

Anti-IFN (neutralizing units)

nmol 2-5A A26o -1 20 h 1 from extracts of cells in

G1 S

0 5.0 50 10 1.7 17

100 1.7 17

affinity [21]. One fraction eluting at a position consistent with that of core yielded an apparent positive result. This, however, was at the limit of detection of the assay and its significance, therefore, is uncertain.

Possible Involvement of Endogenous Interferon (IFN)

As some cell types have been shown to spontaneously produce interferon [9, 27, 28] it seemed possible that changes in the enzyme levels could be related to the presence of interferon secreted into the medium. Supernatants from the cells used for enzyme assays were therefore assessed for activity to inhibit replication of encephalomyocarditis (EMC) virus. Lower levels of activity were found in the G1 quiescent cells, while in cells progressing through the cycle levels rose to an equivalent of approx. 10 IU IFN (fig. 4). The activity was 70 % neutralized by treatment with antibodies to mouse fibroblast IFN and was destroyed by incuba- tion at pH 2, a fact suggesting that an acid-labile a IFN may be involved, but further characterization is needed. It was of interest to determine whether the changes in enzyme levels were related to the presence of endogenous IFN. Cells were therefore treated with antibodies to mouse fibroblast IFN at the G1 quies- cent stage for 15 h and while progressing through the cycle from 3 h until 18 h after seeding (S phase). Table 2 shows that an amount of antibody with capacity to neutralize 10 IU of IFN reduced levels of synthetase in G1 cells by 66% and that the rise from G1 to S was similarly reduced.

Effect of Growth Inhibitor

Since the 2-5A synthetase showed marked changes with cell cycle position, the effect of the inhibitor on the activity of this enzyme during G1 and S phase was also examined. Fig. 5 shows that in cells which had entered S phase treatment for 6 h from 12 to 18 h after seeding caused a 3-fold relative reduction of activity before cell population changes had significantly developed (table 1) and that a reduction of similar extent occurred in G1 cells in response to treatment for l0 h from 2 to 12 h after seeding, i.e. under conditions where no change of population distribution would occur.

Exp Cell Res 159 (1985)

34 Wells and Mallucci

z t

<

10

5

12 18

Time after S e e d i n g (Hours)

Fig. 5. Effect of the fibroblast growth inhibitor on the levels of 2-5A synthetase in extracts from G1 and S phase cells at the tertiary stage expressed as nmol 2-5A synthesized from ATP over a period of 20 h. [], Control cultures; II, cultures treated with growth inhibitor (40 ~tg ml-l) . (Left) Data from cultures harvested at 12 h after seeding; cultures incubated with the inhibitor were treated from 2 to 12 h. (Right) Data from cultures harvested at 18 h after seeding; cultures incubated with the inhibitor were treated from 12 to 18 h.

DISCUSSION

Considerable interest has been focused recently on the possible role of the 2-5A system in cell growth regulation [7-13, 28]. In the work presented here we have analysed the components of the 2-5A system in tertiary cells progressing through one cycle of division. A new approach has been adopted to obtain cell synchrony. Cells of recent derivation have been preferred to immortalized and tumorigenic cells where the pre-replicative period is variable. Also control of synchrony by imposing a seed to yield ratio of 1:2 and by timing changes of nutritional conditions has been preferred to either serum starvation, which favours passage from G1 to GO and further increases pre-replicative differentials [29, 30] or to the use of metabolic inhibitors which perturb the biochemistry of the cell without abolishing G1 differentials after mitosis. The consequence of this novel approach is a cell cycle system where a high degree of uniformity is maintained during each stage of the cycle without altering the physiological state of the cell. Accordingly, in our study, the 2-5A system has been investigated in normal cells undergoing controlled sequential changes while synchronously moving through the cell cycle, rather than by comparing growing and confluent cultures of continuous lines [5, 7, 10] or by using tumorigenic cells [8, 9].

Our results show that the activity of the 2-5A synthetase increased only within a confined period of the cell cycle. Levels were low in quiescent cultures, remained low while cells moved through G1 and the earlier part of S phase, rose rapidly and peaked at about tenfold higher values later in S to decline sharply as cells moved through G2 (fig. 2a). A similar trend was seen for the 2-5A- dependent ribonuclease for which, however, the changes were quantitatively smaller (fig. 2 b). The 2'5' phosphodiesterase activity was lower in cells progress- ing through the cycle than in quiescent cells (fig. 3). These data are in apparent contrast to reports in other systems, where the 2'5' phosphodiesterase has been shown to be high [5] and where 2-5A synthetase and 2-5A-dependent ribonu-

Exp Cell Res 159 (1985)

Cell cycle expression of the 2-5A system 35

clease have been shown to be low [5, 7, 10] in growing cells compared with confluent cells. They also differ from reports where synthetase levels have been shown to decrease when starved cells are stimulated by serum [8, 9]. On the other hand, in accord with our results is the observation that human diploid fibroblasts stimulated by epidermal growth factor (EGF) show an increase in synthetase activity [11]. The reasons for the apparent discrepancies between our results and those of some others [5, 7-10] may relate to the properties of the different cells, and their ability, possibly, to secrete interferon in the growth-arrested state [9, 36]. However, the cell systems per se should also be considered. As discussed above we have investigated normal cells moving through the cycle with a high degree of uniformity rather than comparing growing and confluent cultures where cell cycle position may be difficult to define (exponentially growing cells include multiple populations and confluency does not imply a uniform G1 state) or using tumorigenic cells where unformity within the populations will be impaired by differences in the pre-replicative period.

A point of importance in our system is the production of endogenous interferon (fig. 4, table 2). Levels of interferon low in G1 quiescent cells increased as cells progressed through the cycle could provide an explanation for the synthetase increase in late S phase, a result perhaps consistent with a role of this enzyme in late S phase event. A similar example, where release of endogenous interferon is associated with increase of synthetase activity, has been described in Friend erythroleukemia cells [31].

Despite indications of the presence of phosphorylated 2-5A in crude extracts, analysis by several assays following HPLC fractionation excluded 2-5A down to nM levels in the intact cell. With the assay for core a signal was obtained with material eluting at the appropriate position on HPLC analysis. Although large numbers of cells were used, the signal was too small to be sure of its significance. Hence, even in a situation where interferon is produced and synthetase elevated, no convincing evidence was obtained for the natural occurrence of 2-5A per se or the non-phosphorylated core. This lack of detectable levels is in accord with other reports [4, 32, 33] and need not necessarily suggest that the 2-5A system is not active, as it has been shown that subnanomolar amounts of 2-5A can activate the ribonuclease [35]. It may therefore be concluded that if 2-5A or core is involved in the control of the cell cycle it must be at very low levels or as a result of localized activation of the system [34] or activation for relatively brief periods.

The action of both endogenous interferon and the fibroblast growth inhibitor on our system are worth noting. The increase of endogenous interferon preceded the increase of 2-5A synthetase activity while added inhibitor, on the other hand, acted to reduce levels of the synthetase. These results are of interest as they indicate that an interplay between interferon and the growth inhibitor could be part of the growth-regulatory mechanism of the cell. Experiments have been designed to investigate whether transcription activated by IFN is down-regulated by the inhibitor.

Exp Cell Res 159 (1985)

36 Wells and M a l l u c c i

This work was supported by the Medical Research Council (grant no. G8309413CB). We are deeply grateful to Dr J. Kerr for his interest and involvement in this work. We thank Mr D. Beare for excellent technical help.

R E F E R E N C E S

1. Baglioni, C, Cell 17 (1979) 255. 2. Lengyel, P, Ann rev biochem 51 (1982) 251. 3. Kerr, I M, Humoral factors in host defence p. t41. Academic Press, New York (1983). 4. Silverman, R H, Cayley, B J, Knight, M, Gilbert, C S & Kerr, I M, Eurj biochem 124 (1982) 131. 5. Kimchi, A, Shure, H & Revel, M, Eurj biochem 114 (1981) 5. 6. Jacobson, H, Czasniecki, C, Krause, D, Friedman, R M & Silverman, R H, Virology 125 (1983)

496. 7. Stark, G, Dower, W J, Schimke, T R, Brown, R E & Kerr, I M, Nature 278 (1979) 471. 8. Krishnan, I & Baglioni, C, Mol cell biol 1 (1981) 932. 9. Creasey, A A, Epstein, D A, Marsh, Y V, Khan, Z & Merigan, T, Mol cell biol 3 (1983) 780.

10. Jacobson, H, Krause, D, Friedman, M & Silverman, R, Proc natl acad sci 80 (1983) 4954. 11. Liu, S, T'so, P O P & Hollenberg, M, Life sci 32 (1982) 1479. 12. Kimchi, A, Shure, H & Revel, M, Nature 282 (1979) 849. 13. Kimchi, A, Shure, H, Lapidot, Y, Rapoport, S, Panet, A & Revel, M, FEBS lett 134 (1981) 212. 14. Eppstein, D A, Schryver, B B, Marsh, Y V, Larsen, M A & Kurahara, C G, J interferon res 3

(1983) 305. 15. Taylor-Papadimitriou, J, Interferon vol. 3, p. 14. Academic Press, New York (1980). 16. Wells, V & Mallucci, L, Exp cell res 116 (1978) 301. 17. Mallucci, L, Dunn, M, Wells, V & Delia, D, J cell sci 46 (1980) 353. 18. Mallucci, L, Rasbridge, S & Wells, V, J interferon res 3 (1983) 181. 19. Wells, V & Mallucci, L, J cell physiol 117 (1983) 148. 20. Johannesson, E & Thorell, B, J histochem cytochem 25 (1977) 122. 21. Knight, M, Cayley, P J, Silverman, R I, Wreschner, D H, Gilbert, C S, Brown, R E & Kerr, I M,

Nature 288 (1980) 189. 22. Silverman, R H, Wreschner, D H, Gilbert, C S & Kerr, I M, Eurj biochem 115 (1981) 79. 23. Gresser, I, Tovey, M, Bandu, M, Maury, C & Brouty-Boye, D, J exp med 144 (1976) 1305. 24. Slattery, E, Ghosh, N, Sarnanta, H & Lengyel, P, Proc natl acad sci US 76 (1979) 4778. 25. Wreschner, D H, James, T C, Silverman, R H & Kerr, I M, Nucl acid res 9 (1981) 1571. 26. Hersch, C L, Reid, T R, Friedman, R & Stark, G R, J biol chem 293 (1984) 111. 27. Friedman-Einat, M, Revel, M & Kimchi, A, Mol cell biol 2 (1982) 1472. 28. Revel, M, Kimchi, A, Schulman, L, Wolf, D, Merlin, G, Schmidt, A, Friedman, M, Lapidot, Y &

Rapoport, S, Cellular responses to molecular modulators, p. 361. Academic Press, New York (1981).

29. Baserga, R, Cell tissue kinetics 1 (1976) 167. 30. Augenlicht, L G & Baserga, R, Exp cell res 89 (1974) 255. 31. Revel, M, Kimchi, A, Friedman, M, Wolf, D, Merlin, G, Pouet, A, Rapoport, S & Lapidot, Y,

Texas rept biol med 41 (1981-2) 452. 32. Silverman, R H, Watling, D, Balkwill, F R, Trowsdale, J & Kerr, I M, Eurj biochem 126 (1982)

133. 33. Kerr, I M, Cayley, P J, Roberts, W K, Rice, A, Reid, A, Heish, C, Stark, G R, Laurence, L,

Cailla, H, Marti, J, Wells, V & Mallucci, L, The biology of the interferon system p. 213. Elsevier, Amsterdam (1983).

34. Nilsen, J W & Baglioni, C, Proc natl acad sci US 76 (1979) 2600. 35. Lengyel, P, Interferon (ed I Gressner) vol. 3, p. 78. Academic Press, New York, London (1981).

Vol. 3, p. 78. 36. Friedman-Einat, M, Revel, M & Kimchi, A, Mol cell biol 2 (1982) 1472.

Received October 19, 1984 Revised version received February 8, 1985

Exp Cell Res 159 (1985) Printed in Sweden