Cell, Vol. 6, 253-257,
October
1975,
Copyright
Q 1975
by MIT
The Effects of Topoinhibition Cytochalasin B on Metabolic Michael Stoker Imperial Cancer Research Lincoln’s Inn Fields London WC2, England
Fund
Laboratories
Summary Metabolic cooperation was used to investigate the ability of 3T3 cells to form intercellular junctions under conditions of topoinhibition and topostimulation, respectively. A transformed BHK cell lacking hypoxanthine guanosine phosphoribosyl transferase, which was used as an indicator, was found to incorporate 3H-hypoxanthlne when in prolonged (20 hr) or brief (1 hr) contact with donor 3T3 cells, whether the latter were in a quiescent layer or In the region of stimulation at the edge of a wound. Cytochalasin B (1 yg/ml) prevented the development of metabolic cooperation and abolished most preexisting cooperation between donor 3T3 cells and recipient BHK cells. Cooperation was reestablished by removal of the drug. Cytochalasin B inhibited cooperation by the donor 3T3 cells in both the layer and wound edges, irrespective of topoinhibition and topostimulation. This provides further evidence that alteration In capacity to form stable intercellular junctions is not a necessary feature of the topoinhlbition phenomenon. Introduction When a discontinuity is formed by wounding a stationary monolayer culture of 3T3 cells, growth is stimulated in the cells which migrate from the edge of the residual cell layer into the bare area of the wound (Todaro, Lazar, and Green, 1965; Dulbecco and Stoker, 1970). The failure of the cells in the layer to grow, relative to those in the wound space, has been termed topoinhibition (Dulbecco, 1970). The mechanism of topoinhibition is unknown, but there have been claims that it is due to cell to cell interaction or, alternatively, to local depletion of the medium. In this paper, I shall refer to the opposite, that is, induction of growth at the edge of the wound, as topostimulation. We have recently shown that neither migration of cells away from one another nor alteration of topographical relationships between the cells as seen by light microscopy are essential requisites for topostimulation, because growth still occurs at wound edges of 3T3 cell layers which have been paralyzed by cytochalasin B or by a physical barrier such as a hydrophobic area of substrate (Stoker and Pigott, 1974). There could still, however, be changes in the relationships between cells close to the edge of a layer
and Cooperation which were undetected by ordinary microscopy. For example, there could be an alteration in cell to cell communication, as revealed by low resistance junctions (Loewenstein et al., 1965) or by metabolic cooperation (Subak-Sharpe, Biirk, and Pitts, 1969). In the absence of direct measurements of low resistance junctions or metabolic cooperation directly between the homologous 3T3 cells, I have investigated the ability of 3T3 cells in different parts of wounded cultures to make cooperating junctions with added indicator cells. The system for detection of metabolic cooperation is that originally described by Subak-Sharpe et al. (1969) and further developed by Pitts (1971). A line of polyoma-transformed BHK cells, TGl , lacking hypoxanthine guanine phosphoribosyl transferase (HGRPT) fails to incorporate 3H-labeled hypoxanthine into nucleic acids, and the cells are therefore unlabeled in autoradiographs. 3T3 cells incorporate the purine, and autoradiographs show label over nucleus and cytoplasm. When TGl cells (marked by ingested latex particles) were added to 3T3 cells, those in contact with 3T3 cells became labeled, whereas those that were isolated, or only in contact with other TGl cells, showed no labeling above background. (In confluent monolayers of 3T3 cells, the added TGl cells generally settled into and became part of the monolayer.) Since metabolic cooperation requires active participation and presumably junction formation by both partners (Pitts, 1971), metabolic cooperation between 3T3 cells and TGl cells also implied junction formation between neighboring 3T3 cells. Results Metabolic Cooperation at Wound Edges TGl cells were added 20 hr before or immediately after wounding, and addition of 3H-hypoxanthine or 3H-thymidine, respectively. Table 1 shows the proportion of labeled cells in both types of experiment. Coupling was detected between virtually all the 3T3 and TGl cells in contact, irrespective of the time at which the indicator cells were added, position in relation to the edge, or topostimulation as seen by thymidine incorporation. There was thus no polonged loss of ability to make or maintain cooperating junctions in the stimulated cells. To test for transient loss of cooperation, wounds were made in stationary 3T3 layers to which TGl cells had been added 20 hr previously, and 2 pclml 3H-hypoxanthine were added for 1 or 3 hr before fixation and exposure to film for 6 and 2 days, respectively. Grains were counted in a total of 10 pairs of TGl and 3T3 cells in contact in both the layer and edge. Table 2 shows no obvious change in cooperation up to 9 hr after wounding. The increase
Cell 254
in the wound and decrease in the layer at 3 to 6 hr is of doubtful significance because of irregular distribution of grains over individual ceils.
uptake and cell locomotion (Brownstein et al., 1975). It will also be seen from Figure 1 that the proportion of residual cooperating cells at different doses of cytochalasin B is unaffected by position in the edge or the layer, and is not correlated with topostimulation as shown by thymidine incorporation. Subsequent experiments were carried out with 1 pg/ml of cytochalasin B, which is sufficient to block metabolic cooperation. The drug at this concentration produces some reduction of staining in-
Effect of Cytochalasin B on Metabolic Cooperation Since it has already been shown that topostimulation occurs at wound edges even in cultures paralyzed by cytochalasin B, similar experiments on metabolic cooperation were carried out in the presence of the drug, because delineation of the edge is more precise through absence of cell migration. Unexpectedly, however, the drug was found to block metabolic cooperation. Figure 1 shows the effect of different doses of cytochalasin B on the development of metabolic cooperation between 3T3 and TGl cells. Cooperation was inhibited over a dose range which otherwise only affects glucose
100 TGI CELLS I’,, labelled 3H hypox.
Percentage
(A) 3T3 Cells 3HdT TGl (6)
3T3 Cells “HdT TGl
Edge
October
1975,
Copyright
Q 1975
by MIT
The Effects of Topoinhibition Cytochalasin B on Metabolic Michael Stoker Imperial Cancer Research Lincoln’s Inn Fields London WC2, England
Fund
Laboratories
Summary Metabolic cooperation was used to investigate the ability of 3T3 cells to form intercellular junctions under conditions of topoinhibition and topostimulation, respectively. A transformed BHK cell lacking hypoxanthine guanosine phosphoribosyl transferase, which was used as an indicator, was found to incorporate 3H-hypoxanthlne when in prolonged (20 hr) or brief (1 hr) contact with donor 3T3 cells, whether the latter were in a quiescent layer or In the region of stimulation at the edge of a wound. Cytochalasin B (1 yg/ml) prevented the development of metabolic cooperation and abolished most preexisting cooperation between donor 3T3 cells and recipient BHK cells. Cooperation was reestablished by removal of the drug. Cytochalasin B inhibited cooperation by the donor 3T3 cells in both the layer and wound edges, irrespective of topoinhibition and topostimulation. This provides further evidence that alteration In capacity to form stable intercellular junctions is not a necessary feature of the topoinhlbition phenomenon. Introduction When a discontinuity is formed by wounding a stationary monolayer culture of 3T3 cells, growth is stimulated in the cells which migrate from the edge of the residual cell layer into the bare area of the wound (Todaro, Lazar, and Green, 1965; Dulbecco and Stoker, 1970). The failure of the cells in the layer to grow, relative to those in the wound space, has been termed topoinhibition (Dulbecco, 1970). The mechanism of topoinhibition is unknown, but there have been claims that it is due to cell to cell interaction or, alternatively, to local depletion of the medium. In this paper, I shall refer to the opposite, that is, induction of growth at the edge of the wound, as topostimulation. We have recently shown that neither migration of cells away from one another nor alteration of topographical relationships between the cells as seen by light microscopy are essential requisites for topostimulation, because growth still occurs at wound edges of 3T3 cell layers which have been paralyzed by cytochalasin B or by a physical barrier such as a hydrophobic area of substrate (Stoker and Pigott, 1974). There could still, however, be changes in the relationships between cells close to the edge of a layer
and Cooperation which were undetected by ordinary microscopy. For example, there could be an alteration in cell to cell communication, as revealed by low resistance junctions (Loewenstein et al., 1965) or by metabolic cooperation (Subak-Sharpe, Biirk, and Pitts, 1969). In the absence of direct measurements of low resistance junctions or metabolic cooperation directly between the homologous 3T3 cells, I have investigated the ability of 3T3 cells in different parts of wounded cultures to make cooperating junctions with added indicator cells. The system for detection of metabolic cooperation is that originally described by Subak-Sharpe et al. (1969) and further developed by Pitts (1971). A line of polyoma-transformed BHK cells, TGl , lacking hypoxanthine guanine phosphoribosyl transferase (HGRPT) fails to incorporate 3H-labeled hypoxanthine into nucleic acids, and the cells are therefore unlabeled in autoradiographs. 3T3 cells incorporate the purine, and autoradiographs show label over nucleus and cytoplasm. When TGl cells (marked by ingested latex particles) were added to 3T3 cells, those in contact with 3T3 cells became labeled, whereas those that were isolated, or only in contact with other TGl cells, showed no labeling above background. (In confluent monolayers of 3T3 cells, the added TGl cells generally settled into and became part of the monolayer.) Since metabolic cooperation requires active participation and presumably junction formation by both partners (Pitts, 1971), metabolic cooperation between 3T3 cells and TGl cells also implied junction formation between neighboring 3T3 cells. Results Metabolic Cooperation at Wound Edges TGl cells were added 20 hr before or immediately after wounding, and addition of 3H-hypoxanthine or 3H-thymidine, respectively. Table 1 shows the proportion of labeled cells in both types of experiment. Coupling was detected between virtually all the 3T3 and TGl cells in contact, irrespective of the time at which the indicator cells were added, position in relation to the edge, or topostimulation as seen by thymidine incorporation. There was thus no polonged loss of ability to make or maintain cooperating junctions in the stimulated cells. To test for transient loss of cooperation, wounds were made in stationary 3T3 layers to which TGl cells had been added 20 hr previously, and 2 pclml 3H-hypoxanthine were added for 1 or 3 hr before fixation and exposure to film for 6 and 2 days, respectively. Grains were counted in a total of 10 pairs of TGl and 3T3 cells in contact in both the layer and edge. Table 2 shows no obvious change in cooperation up to 9 hr after wounding. The increase
Cell 254
in the wound and decrease in the layer at 3 to 6 hr is of doubtful significance because of irregular distribution of grains over individual ceils.
uptake and cell locomotion (Brownstein et al., 1975). It will also be seen from Figure 1 that the proportion of residual cooperating cells at different doses of cytochalasin B is unaffected by position in the edge or the layer, and is not correlated with topostimulation as shown by thymidine incorporation. Subsequent experiments were carried out with 1 pg/ml of cytochalasin B, which is sufficient to block metabolic cooperation. The drug at this concentration produces some reduction of staining in-
Effect of Cytochalasin B on Metabolic Cooperation Since it has already been shown that topostimulation occurs at wound edges even in cultures paralyzed by cytochalasin B, similar experiments on metabolic cooperation were carried out in the presence of the drug, because delineation of the edge is more precise through absence of cell migration. Unexpectedly, however, the drug was found to block metabolic cooperation. Figure 1 shows the effect of different doses of cytochalasin B on the development of metabolic cooperation between 3T3 and TGl cells. Cooperation was inhibited over a dose range which otherwise only affects glucose
100 TGI CELLS I’,, labelled 3H hypox.
Percentage
(A) 3T3 Cells 3HdT TGl (6)
3T3 Cells “HdT TGl
Edge
