Bovine fibroblasts transformed by polyoma virus have been cocultivated with syngeneic or allogeneic normal cells. The growth of the transformed cells was inhibited by the presence of normal cells. The strongest depression was obtained when a dense stationary layer of normal fibroblasts was challenged by polyoma cells. The inhibition was least pronounced if normal and transformed cells were simultaneously seeded.

Polyoma-transformed cells were identified by radioautography after incorporation of tritiated thymidine. After 96 h of contact with stationary normal cells the number of grains per nucleus and the number of labelled nuclei had not changed indicating strong depression of DNA synthesis. A deficient attachment or premature detachment of polyoma cells could not account for the observed failure of growth in the normal/polyoma mixed cultures.

The interaction of normal and virus-transformed cells has been studied in tissue culture. In some systems (Stoker, 1964; Stoker, Shearer & O’Neill, 1966; Borek & Sachs, 1966) normal cells inhibit the proliferation of transformed cells. In others (Todaro & Green, 1966; Macintyre & Pontén, 1967) no such effect is manifest. The cause of this difference in the behaviour of transformed cells exposed to normal ones is at present obscure.

The present report forms part of a series where bovine fibroblasts transformed by oncogenic viruses are investigated. The advantage of the system is that stable, well-defined strains of essentially diploid (Lithner & Pontén, 1966) fibroblasts with no tendency to spontaneous transformation even after extended cultivation in homologous serum (Stenkvist & Pontén, 1964; Stenkvist, 1966) are used as controls. These fibroblasts are subject to the three types of growth control—contact inhibition of cell movement (Abercrombie & Heaysman, 1954; Abercrombie, Heaysman & Karthauser, 1957; Stenkvist, 1966), cell cycle inhibition in dense cultures (Eagle, 1965; Macieira-Coelho, 1967; Macintyre & Pontén, 1967) and a finite potential of cell doublings (Hayflick & Moorhead, 1961; Stenkvist, 1966)—regarded as characteristic of normal fibroblasts in vitro. Furthermore, bovine fibroblasts may be transformed by at least four oncogenic viruses—Rous sarcoma virus (RSV) (Stenkvist & Pontén, 1964), Simian virus 40 (Diderholm & Wesslén, 1963; Diderholm, Stenkvist, Pontén & Wesslén, 1965), polyoma virus (Py) (Diderholm, 1967; Thomas & Le Bouvier, 1967) and bovine papilloma virus (Boiron et al. 1964), making comparisons between the effects of different tumour viruses possible within the same system.

The present communication describes the confrontation of normal and polyoma-transformed cells in the same culture. The results show that polyoma-transformed bovine fibroblasts—in contrast to previous findings with RSV-transformed bovine fibroblasts (Macintyre & Pontén, 1967)—are inhibited in the presence of untransformed fibroblasts.

The bovine lung fibroblast strains used—B4 (male), B10 (male), B11 (female), and B13 (female)—were derived from individual embryos. These strains were used as normal untransformed cultures; after transformation by RSV, strain Engelbreth-Holm (Stenkvist & Pontén, 1964)—designated Bio-RSV’, and after transformation by polyoma virus (Diderholm, 1967)—designated B13-Py. We are indebted to Dr Diderholm for the supply of B13 and B13-Py cells. The techniques of cell culture, counting, preparation of chromosomes, cell suspensions and mixtures were directly comparable with those described in a previous communication (Macintyre & Pontén, 1967). The cell counts of the growth curves are the average of two simultaneously harvested plates. From the differences between the duplicate samples a 95 % confidence limit of ± 8·6 % of the mean was calculated for the values plotted in the figures.

The radioautographic techniques were based on those of Doniach & Pele (1950). Specifically, polyoma- or Rous-transformed cells were exposed to tritiated thymidine (3H-TdR, obtained from Schwartz Laboratories with a specific activity of 1·9 c/mM) at a level of 0·01 μc/ml for 7 days. By this procedure 100 % of the nuclei were labelled. The level of radioactivity was sufficiently low to ensure that the growth rate was not impaired. The cells were washed twice with fresh medium, trypsinized, resuspended in Eagle’s MEM containing 1·3 μg/ml of cold thymidine (TdR 10000 × concentration of 3H-TdR) and counted. The suspended, tritiated cells were added in predetermined number to cultures of normal fibroblasts in MEM with 1·3 μg/ml TdR and to dishes without cells. They were maintained in this medium, which was changed three times weekly. Each Petri dish included a 40 × 24 mm coverslip. Coverslips were abstracted at intervals, washed twice in PBS, fixed in methanol: acetic acid (3:1) for 1 h and affixed to a slide with DPX mountant obtained from E. Gurr, 42 Upper Richmond Road West, London, S.W. 14. After dipping in gelatin-chrome alum solution, the slides were covered with Kodak AR 10 stripping film, exposed for 4 days, developed for 10 min in Kodak D-19B, kept in Kodak acid fixer for 10 min, washed for 10 min in running tap water and stained with o-oi % toluidine blue for 5 min. All samples belonging to one experiment were processed simultaneously.

In the ‘Py alone’ or ‘RSV alone’ series all cells on one half of the coverslip were counted under a × 10 objective. In the mixtures between normal and transformed cells, 10 evenly spaced rectangular fields were counted under a × 25 objective, corresponding to one-fifteenth of the total coverslip area.

Nuclear grain counts were made in a predetermined field located in identical position for all coverslips and all labelled cells were examined under oil immersion. The size of the samples is indicated in the table. By multiplying the total number of labelled cells with the average grain count an expression was obtained of the total registered radioactivity per coverslip.

Growth of isolated normal and polyoma-transformed cultures

Growth curves

The growth curve of the normal B13 bovine cells is shown in Fig. 1 and is similar to that for B10 and Bn normals (Macintyre & Pontén, 1967). It should be noted, however, that the B13 cell sheet reached a steady state with pronounced reduction in DNA synthesis (Macieira-Coelho, 1967; E. H. Macintyre, unpublished) and an average mitotic index of < 0·2 % (Macintyre & Pontén, 1967) at a lower density (1 –2 × 108 cells/cm2) than did the B10 or Bn cells (1·8 × 105 cells/cm2). The growth curves for the polyoma-transformed cultures differed from control curves. The rate of growth was significantly faster and higher cell densities were attained. Within the experimental period of 30 days, maxima of 10·2 × 108 and 9 × 106 cells per cm2 were attained by the 108 and 104 initial seedings of Py cells respectively. Mitotic activity was still high even at this level and no cell density—dependent inhibition of the cell cycle akin to that observed in the untransformed cultures (E. H. Macintyre, unpublished) could be demonstrated. The ‘steady state’ approached by the Py cells thus reflected a balance between cell death and proliferation rather than inhibition of mitosis.

Fig. 1.

Growth curves for untransformed bovine fibroblasts and the same cells transformed by polyoma virus. The individual points represent averages between duplicate dishes.

Fig. 1.

Growth curves for untransformed bovine fibroblasts and the same cells transformed by polyoma virus. The individual points represent averages between duplicate dishes.

Morphology

Polyoma-transformed cultures consisted of overlapping, randomly oriented fibroblast-like elements in contrast to the regular layering characteristic of untransformed cultures. The arrangement of the Py cells differed from that of RSV-transformed cells mainly in the absence of dense piles of cells.

Growth of mixed cultures of untransformed and transformed cells

The first three experiments were designed to parallel those already reported for the normal/Rous-transformed mixtures (Macintyre & Pontén, 1967). Transformed cells were added to normal ones, which were (a) simultaneously seeded, (b) in logarithmic, or (c) post logarithmic phase.

Simultaneous seeding of normal B11 and Py-transformed B13 cells (Fig. 2)

Five experimental groups were set up. These were 700000 B13/100000 B11/ 700000 Bn/ 100000 B13-Py, 700000 B11/20000 B13-Py, 100000 Big-Py and 20000 B13-Py.

Fig. 2.

Growth curves for polyoma cells (line B13-Py) seeded simultaneously with normal bovine fibroblasts (line B11) on day o. 700000 normal fibroblasts were mixed with 100000 or 20000 B13-Py. The two broken lines represent total plate counts minus cell counts in the control group (B11/B13) expressed as cell densities × 106. The unbroken lines represent cell densities in the mixture of normal cells (B11/B13) or among B13-Py grown in isolation. Vertical bars indicate the 95 % confidence limits.

Fig. 2.

Growth curves for polyoma cells (line B13-Py) seeded simultaneously with normal bovine fibroblasts (line B11) on day o. 700000 normal fibroblasts were mixed with 100000 or 20000 B13-Py. The two broken lines represent total plate counts minus cell counts in the control group (B11/B13) expressed as cell densities × 106. The unbroken lines represent cell densities in the mixture of normal cells (B11/B13) or among B13-Py grown in isolation. Vertical bars indicate the 95 % confidence limits.

The mixture of untransformed fibroblasts stabilized by day 11 at 1 –8 ×105 cells per cm2, which corresponds to the level previously established for B11 cultures (Macintyre & Pontén, 1967). 108 or 2 × 104Py cells grown in the absence of untransformed fibroblasts increased continuously in number during the 18 days the experiment lasted to reach 6·7 and 4·0 ×105 cells per cm2, respectively. The same number of Py cells seeded simultaneously with control fibroblasts failed to show the rise in cell number expected if no inhibition were present. Not until day 11 and day 18 was there a small increase in the computed number of Py cells. This increment was, however, considerably less than the value expected under conditions of unrestrained multiplication. For example, on day 18, 100000 Py cells grown in isolation had reached 6·7 × 105 cells per cm2, whereas the same number of Py cells seeded together with normal cells only rose to a computed number of 2·0 × 108 cells per cm2.

Addition of Py-transformed cells to exponentially growing normal fibroblasts

Of many experiments giving the same results, we will consider two in detail, where untransformed B13 cells were seeded at 500000 on day o. One experiment comprised 5 experimental groups set up on day two: B13/10000 B13; B13/500 B13-Py; B13/10000 B13-Py, 500 B13-Py and 10000 B13-Py. Figure 3 depicts the information obtained from cell counts on the 10000 Py-cell group from day 3, the day after mixture. The dotted line, as in Fig. 2, represents the numerical difference between the mixture of normal and transformed cells and the corresponding normal control count. A small, but statistically significant excess of 250000 cells was found on day 11, which rose to 0·5 × 106 cells per cm2 by day 18 and to 0·9 × 106 cells per cm2 by day 25.

Fig. 3.

Growth curves for polyoma-transformed cells added to multiplying syngeneic normal fibroblasts. 500000 normal fibroblasts (B13) were seeded on day o. On day 2, 10000 B13 and B13-Py cells were added to the previously seeded fibroblasts and 10000 B13-Py seeded into empty dishes. The unbroken lines represent cell densities; the broken line total plate counts minus the respective counts for the normal control cells also expressed as cell densities. Vertical bars indicate 95 % confidence limits.

Fig. 3.

Growth curves for polyoma-transformed cells added to multiplying syngeneic normal fibroblasts. 500000 normal fibroblasts (B13) were seeded on day o. On day 2, 10000 B13 and B13-Py cells were added to the previously seeded fibroblasts and 10000 B13-Py seeded into empty dishes. The unbroken lines represent cell densities; the broken line total plate counts minus the respective counts for the normal control cells also expressed as cell densities. Vertical bars indicate 95 % confidence limits.

A further variation was tried, when 260000 B13-Py cells were added to B13 cells in late growth phase (Fig. 4), 7 days after seeding of B13. Despite the high numbers of transformed cells added, there was no significant cell growth in these plates, a finding comparable to those depicted in Fig. 3.

Fig. 4.

Growth curves for polyoma-transformed cells added to multiplying syngeneic normal fibroblasts. 500000 normal fibroblasts (B13) were seeded on day o. On day 7, 260000 B13 and B13-Py were added to the previously seeded fibroblasts and the same number of B13—Py seeded into empty dishes. The unbroken lines represent densities; the broken line total plate counts minus the respective counts for the normal control cells also expressed as cell densities. Vertical bars indicate 95 % confidence limits.

Fig. 4.

Growth curves for polyoma-transformed cells added to multiplying syngeneic normal fibroblasts. 500000 normal fibroblasts (B13) were seeded on day o. On day 7, 260000 B13 and B13-Py were added to the previously seeded fibroblasts and the same number of B13—Py seeded into empty dishes. The unbroken lines represent densities; the broken line total plate counts minus the respective counts for the normal control cells also expressed as cell densities. Vertical bars indicate 95 % confidence limits.

Addition of transformed cells to normal cells in stationary post-exponential growth phase (Fig. 5)

Stationary monolayers of B13 normal fibroblasts were allowed to form after seeding approximately 700000 cells on day o. Challenge normal and transformed B13-Py cells were added on day 12. Five experimental groups were set up—B13/ 10000 B13; B13/10000 B13-Py, B13/500 B13-Py, 10000 B13-Py and 500 B13-Py. The experiment was carried for 15 more days, i.e. until day 27 after seeding of the normal base layer. The B13/B13 maintained a steady cell count, and the mitotic indices varied only between 0·0 and 0·4 %. As seen from Fig. 5 there was no increase in the computed cell number among the admixed Py cells until day 25 or day 27 when a small rise was recorded. Py cells grown by themselves showed the expected high growth rate and failed to reach a stable level within the experimental period.

Fig. 5.

Growth curves for polyoma-transformed cells added to stationary layers of syngeneic normal cells. On day 12, 10000 or 500 Py cells were added to stationary layers of untransformed fibroblasts. Simultaneously the same number of Py cells were seeded into empty dishes. Unbroken lines represent cell densities; broken lines total plate counts minus the respective counts for the normal controls expressed also as cell densities. Vertical bars indicate 95 % confidence limits.

Fig. 5.

Growth curves for polyoma-transformed cells added to stationary layers of syngeneic normal cells. On day 12, 10000 or 500 Py cells were added to stationary layers of untransformed fibroblasts. Simultaneously the same number of Py cells were seeded into empty dishes. Unbroken lines represent cell densities; broken lines total plate counts minus the respective counts for the normal controls expressed also as cell densities. Vertical bars indicate 95 % confidence limits.

In the same experiment the capacity of the stationary B13 monolayer to permit growth of RSV-transformed bovine fibroblasts (B10-RSV) was tested. In accordance with previous findings (Macintyre & Pontén, 1967) no inhibition of Rous cell proliferation was noted when 500 B10-RSV or 150000 B10-RSV cells were added to the stationary B13 monolayer (not illustrated).

Addition of transformed cells to normal stationary cells in late phase II of growth

The apparent inhibition of growth exerted by stationary normal bovine fibroblasts on polyoma-transformed cells could be an age-dependent phenomenon and not manifested in phase III, i.e. during the terminal degenerative stage when normal fibroblasts approach the end of their finite life span in vitro (Hayflick & Moorhead, 1961). This possibility was tested by adding polyoma cells to stationary B4 fibroblasts which were at transfer 65 and therefore in early phase III (Stenkvist, 1966). The polyoma cells, at passage number 33, represented phase II cells. In this phase of growth, cells proliferate vigorously in culture. Table 1 shows that inhibition of polyoma cells was also exerted by normal bovine cells in early phase III.

Table 1.

Ability of late passage normal bovine fibroblasts to inhibit proliferation of polyoma-transformed bovine cells

Ability of late passage normal bovine fibroblasts to inhibit proliferation of polyoma-transformed bovine cells
Ability of late passage normal bovine fibroblasts to inhibit proliferation of polyoma-transformed bovine cells

Cell mixtures—radioautographic studies

Prelabelled B13-Py or B13-RSV cells were added to empty 50-mm plastic dishes and to dishes containing stationary layers of normal B10 or B13 cells. Coverslips were abstracted at different intervals and analysed for total number of labelled cells and number of grains per labelled nucleus.

It is seen (Table 2) that in all 4 groups the number of labelled cells was lower than the inoculated number shortly after seeding. In the two groups with RSV cells this lag phase lasted between 10 and 18 h, in the ‘Py alone’ an increase in cell number was not recorded until 48 h. Py cells on top of normal fibroblasts failed to exceed the inoculated cell number during the entire period of observation, as could be expected from the results shown in Fig. 5.

Table 2.

Radioautographic analysis of proliferation of polyoma and RSV-transformed bovine fibroblasts

Radioautographic analysis of proliferation of polyoma and RSV-transformed bovine fibroblasts
Radioautographic analysis of proliferation of polyoma and RSV-transformed bovine fibroblasts

The recorded growth rate of the labelled transformed cells was in the same range as that of the corresponding unlabelled cells (Fig. 1; Macintyre & Pontén, 1967).

The number of grains per nucleus decreased in the ‘Py alone’, the ‘RSV alone’ and ‘normal fibroblasts plus RSV-cells’ groups. The decrease was inversely proportional to the cell count, and the total number of grains thus remained essentially constant. This shows that no major detachment of cells occurred. The proliferation rate of normal/Rous mixtures exceeded those of Rous cells alone, further supporting that normal cells act as ‘feeders’ in this system (Macintyre & Pontén, 1967).

In contrast to the three other groups the Py cells seeded on normal fibroblasts failed to show any significant decrease in the average number of grains per nucleus. Since the total grain count also remained constant, the Py cells must have remained attached with a repressed cell cycle. The proportion of cells attaching seemed to be lower when Py cells were seeded on to normal fibroblasts rather than bare plastic.

Inhibition of polyoma-transformed rodent cells by untransformed cells has been described previously (Stoker, 1964; Stoker et al. 1966; Borek & Sachs, 1966). The present data show that the same effects are obtained with transformed bovine cells. This firmly supports the conclusion that the inhibition is connected with polyoma virus infection and not dependent on any peculiar properties of rodent cells.

Strong inhibition of Py-cell growth was recorded in autologous and homologous mixtures. Growth of RSV-transformed cells was previously shown to be uninhibited regardless of whether homologous or autologous normal fibroblasts were used (Macintyre & Pontén, 1967). Allogeneic differences therefore do not seem to play a decisive role in the bovine system. The lack of a suitable in vivo system has precluded the determination of the eventual presence of transplantation antigens on our transformed Py and RSV cells. The presence of such antigens has been suggested as an explanation for the inhibitions observed in various mixtures of normal and transformed hamster cells (Borek & Sachs, 1966). On the basis of evidence from other mammalian systems (Jonsson & Sjogren, 1965), RSV would be expected to induce transplantation antigens also in bovine cells. If this should be true, the absence of inhibition of RSV cells would speak against transplantation antigens being determinants for cell-density-dependent inhibition of the division cycle. The continuing inhibition by normal fibroblasts in both early and late phase II growth phase of polyoma cells indicates that the phenomenon is not culture-age-dependent.

The radioautographic marker used here has certain advantages over previously employed systems where phagocytosed dye particles were used. As pointed out by Stoker (1967) two different dyes have to be employed and the results can even then be evaluated only on a statistical basis. The 3H-TdR level was not high enough to interfere with cell proliferation in either the polyoma- or Rous-transformed cells. The present use of transformed cells whose DNA had been lightly labelled with tritiated thymidine permitted a rather detailed analysis of the events after seeding of RSV- and polyoma-transformed cells. The depression of cell proliferation in the mixed cultures of normal and Py-transformed fibroblasts was accompanied by inhibition of DNA synthesis at least during the first 96 h and could not be ascribed to a deficient attachment or rapid detachment of the polyoma cells.

The inhibition of polyoma cells as judged from the cell counts was observed over a considerable length of time in contrast to previous investigations (Stoker, 1964; Stoker et al. 1966; Borek & Sachs, 1966) which concentrated on shorter terms of observation. When stationary fibroblasts were used as a base, 11 days elapsed before any proliferation ascribable to the polyoma cells was recorded. The reasons for this eventual breakthrough may be trivial and due to partial detachment of the base layer which might permit polyoma cells to establish themselves as isolated clusters. Cells placed centrally in such heaps would then be spatially separated from the inhibitory influence of the normal cells.

The physical and chemical basis for cell cycle inhibition is largely unknown. Stoker (1964) has suggested the existence of an efferent (emitter) and an afferent (receptor) link, which only interact at very short distances and thus depend on intimate cell-cell contacts. Both are assumed to be present among normal fibroblasts and are responsible for the cell cycle inhibition readily observed when such cells have established intimate contacts (Levine, Becker, Boone & Eagle, 1965; Macieira-Coelho, Pontén & Philipson, 1966). Since the polyoma transformed cells do not inhibit each other but are inhibited by normal cells, they may be assumed to lack emitter but retain receptor. RSV cells, on the other hand, are not inhibited and should accordingly lack receptor. Whether this lack is an isolated defect or combined with lack of emitter is undecided.

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