A cell cycle study of the effects of Con A on synchronized mouse embryo fibroblasts: arrest at two specific stages of the cycle and dissociation between uptake of thymidine and DNA synthesis

The lectin Concanavalin A (Con A) binds to specific determinants of the cell surface (Allen, Auger & Crumpton, 1972) and like other ligands can induce structural and functional changes of the plasma membrane. Structural changes include redistribution of macromolecular components within the plane of the membrane brought about by the cross-linking properties of the tetrameric lectin molecule (De Petris, Raff & Mallucci, 1973; Raff, De Petris & Mallucci, 1974; Nicolson, 1974) and the alteration of an association with the internal acto-myosin system (Ash & Singer, 1976; Toh & Hard, 1977; Lilly, Bourgignon & Singer, 1977). Functional changes include modification of active transport functions (Van den Berg & Betel, 1971; Peters & Hausen, 1971; Van den Berg& Betel, 1973).

Con A has also an effect on cell growth; it can induce blast transformation and mitosis in normal lymphocytes (Powell & Sachs, 1970; Novogrodsky & Katchalski, 1971; Andersson, Moller & Sjoberg, 1972; Gunther, Wang & Edelman, 1974) while at higher doses it can inhibit growth of both lymphocytes and proliferating lymphoid cells (Mackler, 1972; Ralph & Nakoinz, 1973; McClain & Edelman, 1976). A recent analysis of DNA replication in S-phase lymphocytes has shown that replicon initiation is inhibited by high doses of Con A but that DNA elongation is not affected (Lanotte & Moerman, 1979).

In cells of fibroblastic derivation doses of Con A higher than those inducing blast transformation inhibit the re-induction of thymidine uptake by serum stimulation in chick embryo cells that have reached confluence (McClain, Eustachio & Edelman, 1977) and it has been proposed that Con A arrests cells at a definite point of the G₁ stage (McClain & Edelman, 1976; McClain et al. 1977). Uptake of thymidine, however, may not accurately reflect the DNA replication which is actually taking place if an alteration of the relationship between surface membrane and the acto-myosin system is induced by the lectin, as it has been shown that cytochalasin B, which affects actin polymerization (Lin & Lin, 1979; Grumet, Flanagan, Lin & Lin, 1979), causes inhibition of thymidine uptake while DNA synthesis remains unaffected (Everhart & Rubin, 1974; Brownstein, Rozengurt, Jimenez de Asua & Stoker, 1975). Also, due to its ability to induce redistribution of surface macromolecules within the plane of the membrane, Con A could directly affect membrane functions concerned with the initial steps of nucleoside uptake (Plagemann & Richey, 1974; Plagemann, Richey, Zylka & Erbe, 1975).

In the present cell cycle study we have used a fluorimetric method to assess cellular DNA content and have examined the effect caused by Con A on cell growth and on DNA synthesis and thymidine uptake. We found that Con A did not solely prevent cell entry into S-phase (arrest in G₁) but that it also arrested S-phase cells in G₂ and prevented G₂ cells from dividing, thus causing a G₂ block. We also found a dissociation between DNA synthesis, which continued, and thymidine uptake, which was inhibited, in cells exposed to Con A while progressing through S-phase. The inhibition of uptake was greater when Con A receptors were extensively clustered.

Cultures of synchronized tertiary mouse embryo fibroblasts were derived from C57 Bl mice. Synchrony was obtained without the use of metabolic inhibitors, controlling cell growth by varying the cultural conditions throughout the primary and secondary cultivations as previously described (Wells & Mallucci, 1978). Both for growth experiments and radioactive labelling the tertiary fibroblasts were seeded in scintillation vials at a density (1·2 x 10⁵ cells cm⁻²) approxi mately equal to half the density expected at final confluence using Eagle’s BHK medium supplemented with 10 % tryptose phosphate broth and 5 % foetal bovine serum and containing 0·1 % sodium bicarbonate. Cultures were equilibrated with 5 % CO₂ in air and incubated in a 37 °C waterbath.

Con A 3 times crystallized (Miles Laboratories Ltd) was dissolved in 0·15 M phosphate-buffered saline pH 7·3 (PBS) at the required concentration immediately prior to use. The dimeric succinylated derivative (succinyl-Con A) was prepared according to the method of Gunther et al. (1973), freeze-dried and dissolved in PBS when required. Control cultures received equivalent volumes of PBS. Binding between lectins and substrate was reversed using the competing sugar a-methyl-mannoside (Goldstein, 1976). Lectin preparations were used at doses of 50-100 µg ml⁻¹ which did not affect cell viability as assessed by trypan blue exclusion.

Cellular DNA content was quantitated using a fluorescent activated cell sorter (Johannesson & Thorell, 1977) and the percentage of cells occupying different stages of the cycle assessed at various intervals throughout the duration of one division cycle of the synchronous population. The cultures were treated with 0·05 M a-methyl-mannoside in order to facilitate their removal from the glass and were exposed to 1: 5000 versene solution containing 0·1 % trypsin. After neutralization the cells were centrifuged and washed twice with PBS at 4 °C, fi.xed in 70 % ethanol in PBS, and then resuspended in 20 µg ml⁻¹ mithramycin in 25 % ethanol containing 15 mM MgCl₂. Cell number was assessed using a Coulter counter.

Cultures in scintillation vials were incubated in a 37 °C waterbath and pulse-labelled for various times with 0·1 µM [³H]thymidine ([Me⁻³H]thymidine, 18-25 Ci mmol-1, Radio chemical Centre, Amersham). Uptake into acid-precipitable and acid-soluble fractions was assessed as previously described (Wells & Mallucci, 1978). For Lineweaver-Burk analysis cells were incubated for 5 min with concentrations of thymidine ranging from 0·05 to 0·5 μM and total uptake recorded.

This method has been described in detail elsewhere (Mallucci, 1971, 1976). Briefly, syn chronized tertiary fibroblasts were grown on coverslip vials (Mallucci, 1976) incubated in a 37 °C waterbath. The cultures were treated with lecrins and fixed with 2·5 % glutaraldehyde in PBS for 1 h. Indirect immunofluorescence staining was carried out using the IgG fraction purified from an antiserum to Con A and an FITC-conjugated goat anti-rabbit serum. The cells were examined by Nomarski optics and by epi-illumination using energy in the blue light range.

The experiment reported in Fig. 1 shows the relative distribution of cells occupying the G₁, S and G₂ stages of the cycle, as assessed by fluorimetric measurements of DNA content at 24 and 32 h after seeding. As expected from previous cell kinetic studies (Wells & Mallucci, 1978) control cells (Fig. 1A) reached the G₂ stage by 24 h and divided by 32 h, returning to a pattern similar to that of the original seed. When cells were treated with 50 µg ml⁻¹ of Con A while in G₁ they did not significantly alter from the original distribution of the seed (Fig. 1B). On the other hand, in cells treated while progressing through S-phase the peak representing the G₂ population increased from 24 to 32 h.

A more detailed analysis of the S-G₂ distribution throughout the cycle in cells treated with Con A during G₁, during S and after completion of Sis reported in Fig. 2. In the absence of lectin the synchronized cells entered S-phase at about 12 h and progressed through DNA synthesis and division completing their cycle in about 32 h. When Con A was added at 8 h after seeding, DNA synthesis was, as expected, pre vented and the cells remained in the G₁–G₀ stage as shown by a lack of significant increase in the percentage of the S-G₂ population. When Con A was added during DNA replication, at 17 h after seeding, cellular DNA content continued to increase in parallel with that of controls, but the cells did not divide. Instead they entered and remained in the G₂ stage. Cell division was also prevented and cells did not cross G₂ when Con A was added at 23 h, a time when DNA replication had reached or neared completion. Thus, once initiated, the process of DNA replication itself was not affected but Con A inhibited growth either by preventing cells from leaving the G_cG₀ stage or by arresting them in G₂

The data of Table 1 show that growth inhibition was not merely due to a cytotoxic effect exerted by Con A, although some degree of toxicity did occur, particularly in cells treated at the S and the G₂ stages of the cycle, as in all cases the inhibition was reversed and cells divided after addition of the competing sugar a-methyl-mannoside.

Studies based on the assessment of thymidine uptake have led to the conclusion that Con A can inhibit cellular DNA synthesis (Mackler, 1972; Ralph & Nakoinz, 1973) but it has been shown that although initiation of DNA synthesis is prevented by Con A, chain elongation is not (Lanotte & Moerman, 1979). The results presented in the previous section also demonstrate that Con A added during S-phase does not alter the rate of increase of cellular DNA content. We have therefore investigated whether in our system thymidine uptake was affected by Con A, and if so, whether it was the ability of the tetrameric lectin molecule to cross-link and induce redistribution of surface macromolecules that could be responsible for any variations in the pattern of uptake. In these experiments succinyl-Con A, the dimeric form of the lectin which binds to the same cell surface determinants but has less cross-linking ability (Wang, Gunther & Edelman, 1976), was also used. Lectins were added to cells in S-phase at 17 h after seeding, a time when they were nearing the peak of DNA synthesis. The data of Fig. 3 show that neither form of lectin had any immediate effect but that after an incubation period of 2 h there was a marked reduction in uptake when Con A was present but only a marginal effect in the presence of succinyl-Con A. A Lineweaver Burk estimation carried out using the initial 5-min measurements showed that Con A had no effect on Km but caused a 30 % decrease of the relative V_max value as expressed by uptake of radioactivity.

The lag time required for the establishment of the Con A effect and the inability of succinyl-Con A to establish one, prompted us to use specific immunofluorescence to investigate the pattern of distribution on the cell surface of both forms of the lectin. Fig. 4 shows well defined domains of fluorescence at a time when the uptake process had been markedly reduced but a fairly even distribution where no significant changes had, or had yet, taken place.

Our results demonstrate that Con A arrested cells both in the GcG₀ and in the G₂ stages but not in S-phase and that Con A inhibited the S-phase-associated process of thymidine uptake without affecting macromolecular synthesis. A dissociation between uptake of thymidine and DNA synthesis of similar type to the one induced by Con A described here has been observed under conditions where the cytoskeleton is interfered with, such as in the case of cytochalasin B treatment of Chinese hamster ovary cells (Everhart & Rubin, 1974) and of 3T3 cells stimulated by serum (Brownstein et al. 1975), and after blastogenic response to Con A in the presence of tubule-disrupting drugs (Steen & Lindmo, 1978). The same type of dissociation has also been described in leukaemic 1210 cells (Brouty-Boyé & Tovey, 1977) and in mouse fibroblasts (Mal lucci, Dunn & Wells, 1980) exposed to interferon. Our data also show that the reduction of uptake was of a quantitative rather than a qualitative type and that it coincided with clustering of cell-surface receptors, thus suggesting that the operational state of membrane function(s) concerned with the early stages of nucleoside uptake had been quenched in parts following the extensive cross-linking of Con A receptors. Although Con A prevented the commencement of DNA synthesis by preventing quiescent cells from entering S-phase, Con A did not affect DNA replication in cells that had advanced into S-phase as further progression through S was not altered. These results are in accord with the previous reports of McClain et al. (1977) and of Lanotte & Moerman (1979). Our data, however, also show that Con A caused S-phase cells to arrest in G₂ and cells that had already reached this stage to remain there, thus preventing division.

The fact that cells were arrested both at the G₁–G₀ and the G₂ stages of the cycle is of interest as it indicates the existence of 2 key points in the cell cycle on which Con A can exert its action. The underlying condition at these points is not known but the fact that Con A can affect the distribution of surface macromolecules and that functional associations between surface macromolecules and the cytokinetic elements have been described in numerous systems (Ash & Singer, 1976; Toh & Hard, 1977; Sundqvist & Ehrnst, 1976; Lilly et al. 1977; Ash, Louvard & Singer, 1977; Gabbiani, Chaponnier, Zumbe & Vassalli, 1977; Schreiner, Fujiwara, Pollard & Unanue, 1977; Koch& Smith, 1978; Flanagan & Koch, 1978; Thom, Cox, Sefford & Rees, 1979; Hoesli, Rungger Brandle, Jockusch & Gabbiani, 1980) leads to the suggestion that during these 2 periods of the cycle there is a critical relationship between macromolecular components of the plasma membrane which can be rearranged by Con A and the operational state of the cytokinetic system. Such a relationship is immediately relevant when the contractile apparatus is about to be formed in preparation for mitosis (late G₂), but the interplay between surface and contractile elements when cells prepare to leave the quiescent state may also be important, and our experiments indicate that the Con A block on cell growth operates at these two moments. In previous work (Wells & Mallucci, 1978) we have shown that the topographical distribution of cell surface macromolecules affects the operational stability of the microfilament network and the redistribution of the cytoplasmic mass resulting in changes of cell shape and that the functional cooperation between surface macromolecules and contractile elements can be altered by Con A.

This work was supported by the Cancer Research Campaign. We thank Dr E. Rozengurt for helpful criticism.