- Email: [email protected]
Cell exudation and cell adhesion
Experimental Cell Research 71 (1972) 204-208
CELL EXUDATION
AND CELL ADHESION
D. E. MASLOW and L. WEISS Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, N. Y. 14203, USA
SUMMARY Experiments are described in which the exudation of 51Cr-labelled material from fibroblasts and Ehrlich ascites tumor (EAT) cells was correlated with their adhesion to cellular and noncellular surfaces, in vitro. A correlation coefficient of greater than + 0.90 was obtained for adhesion of cells to glass and the release associated with it for both cell types. The release per cell for EAT cells adhering to a fibroblast monolayer was greater than for a glass substrate. Fibroblasts showed no significant difference between adherence to glass or to a fibroblast monolayer. The apparent “substratum specificity” of Vr-release exhibited by the EAT cells, and its apparent loss by the fibroblasts is discussed.
In spite of intensive investigations, the detailed mechanisms of cell adhesion remain obscure. Although cellular exudation has been studied as one of the consequences of cell adhesion [14, 18, 271 its role in promoting cell adhesion has received comparatively little attention. In this communication we describe experiments in which the exudation of 51Cr-labelled material from cells was correlated with their adhesion to cellular and non-cellular surfaces, in vitro. MATERIAL
AND METHODS
Cell culture All cell culture medium and reagents, unless otherwise indicated, were obtained from Microbiological Associates (Bethesda, Md). Cells: (a) Fibroblasts-mouse fibroblasts of the L-929 line are maintained routinely in this laboratory on glass at 37”C, in RPM1 1640 medium [9] supplemented with 20% fetal bovine serum, in an atmosphere of 5 % CO2 in air. They were freed from glass by incubation in 0.2 % trypsin in solution, for 3 min at 37°C. (b) Ehrlich ascites tumor cells-EAT cells of the Exptl Cell Res 71
Lettre hyperdiploid strain are maintained in suspension culture in RPM1 1630 medium [lo] supplemented with 5 % calf serum. Culture vessels: Culture vessels were made by sticking Teflon rings of 1.3 cm inside diameter and 0.3 cm height, to- cleaned glass slides or coverslips, with either silicone grease or paraffin. Monolayers were obtained by adding 0.25 ml of cell suspension at a concentration of approx. 600000 cells/ml, to the chambers and incubating at 37°C for up to 24 h in a 5 % CO,-in air, high humidity atmosphere. Suspensions of labelled cells were added at concentrations of 300000 cells/ml to vessels, with or without monolayers. Labelling: Tr: Na 51CrOa, equivalent to 25 ,uCi activity, was added to 10 ml of cell suspension containing’approx. 10’ cells. After 30 min incubation at 37°C. the cells were washed with cold medium and kept at 4°C for 4 h. They were used after 2 additional washes in medium. At intervals after the addition of the cells to the chambers, the medium was removed and the suspended cells spun down in a Coleman microcentrifuge for 1 min. The supematant was removed and counted, as was the cell pellet and the coverslip, in a Packard Auto-Gamma counter. 8H: Ten /Xi of *H-thymidine was added to cultures of fibroblasts 6 h after seeding, and at 24 and 48 h thereafter. After an additional 24 h. the cells were resuspended and washed 3 times in ‘medium before use. EAT cells were incubated for 1 h with 5 &i of 8H-uridine in 5 ml of medium containing approx. 20 x 10’ cells, and used after 3 washes.
Cell exudation and cell adhesion
205
-6
in fig. 1. The correlation coefficient for the data on cell adherence and release of radioactive chromium for the typical experiment shown is +0.933. The percentage release of 51Cr in a gently agitated suspension of EAT cells after 90 min was 12.6%.
-3
Adherence of jibroblzts
-9
Fig. 1. Abscissa: time (min); ordinate: (left) O-0, % released 61Cr in the sunematant fraction: (right) O-O, average number of cells/field. EAT cell adherence and 51Cr release after settling onto a glass substrate in a typical experiment. Autoradiography: Formalin-fixed preparations were stained with Mayer hematoxylin, coated with Kodak Nuclear Track Emulsion-NTB 3, and developed after 2 weeks. Counts of adherent cells: After gently rinsing the coverslips free of non-adherent cells, with serum-free medium, the adherent cells were counted. Routinely, the cells adhering to glass were counted bv nhasecontrast microsc
to glass
The number of fibroblasts adherent to glass, increased with time during the 100 min duration of the experiments (fig. 2). During the same time period, the percentage release of 51Crincreased up to approx. 70 and 90% in the two experiments noted in fig. 2. The correlation coefficient for these adhesion and 51Cr-releasedata was greater than +0.90 in all of the experiments. The percentages of released radioactivity in the suspensions of fibroblasts prevented from adhering were 23.8 and 28.2 respectively. Adherence of EAT cells to jibroblast monolayers
The number of EAT cells adherent to fibroblast monolayers increased up to 60 min
RESULTS Adherence of EAT cells to glass
The number of EAT cells that remained adherent to glass increased with time up to 60 min. Thereafter, the number did not change significantly (fig. 1). The release of 51Cr,as measured by the percentage of counts in the supernatant fraction of that in the final cell suspension, also increased progressively up to 60 min, after which no significant increase was noted in the cultures, as shown
Fig. 2. Abscissa: time (mm); ordinate: (left) e--a, average number of cells/field; (rinht) O-O, A-A, percentage of released %r in the sup&natant fraction: Fibroblast cell adherence and 61Cr release after settling onto a glass substrate in some typical experiments. Exptl Cell Res 71
206
D. E. Maslow & L. Weiss
in culture, but not subsequently. The percentage release of 51Cr, however, increased throughout the 90 min course of these experiments. Experiments were also made in which the percentage release of 51Cr, or the number of adherent cells, was determined for simultaneous cultures of EAT cells interacting with glass or fibroblast monolayers. In these two groups of experiments, no significant differences were found between the numbers of EAT cells adherent to the glass or fibroblasts; however, the average percentage release of 51Cr was significantly greater in the case of cells adhering to the monolayer as shown in fig. 3. Comparison of the mean percentages of 51Cr released by standard t-tests, revealed that at all times studied, 0.05 >p >O.Ol. Eighteen and 19 % of 51Cr was released from the EAT cells used in two of these groups of experiments, after 90 min in suspension culture, when adhesion was prevented by gentle agitation. The adherence of fibroblast cells to a fibroblast monolayer
The numbers of fibroblast cells adhering to a fibroblast monolayer increased throughout I 60 1
40
///A0 /
I
20
I ,’ 0
60
40
0
0
30
60
9C
Fig. 4. Abscissa: time (min); ordinate: average percentage of released Wr in the supematant fraction; (left) O-O, fibroblast monolayer substrate; (right) O-O, glass substrate. Comnarison of the release of Wr from fibroblast cells settling onto a fibroblast monolayer and glass.
the 90 min course of these experiments. The numbers of cells that adhered to the monolayer were not significantly different from those adhering to a glass surface in control cultures. The releaseof the 51Crfrom the fibroblasts, adhering to a fibroblast monolayer also increased during the 90 min observed (fig. 4). The correlation coefficient for the increase in release and increase in adherent cells was greater than +0.90 in all the experiments. The percentage release of 51Crwas not significantly different (p=O.4 to 0.9) when the fibroblasts adhered to glass or to a fibroblast monolayer (fig. 4).
1
! L
r/ 01
80-
I 30
I 60
I I 90
Fig. 3. Abscissa: time (min); ordinate: average percentage of released 61Cr in the supematant fraction; (left) O-O, fibroblast monolayer substrate; (right) o-o, glass substrate. Comparison of the release of Wr from EAT cells settling onto a fibroblast monolayer and glass. Exptl Cell Res 71
DISCUSSION In recent years, much attention has been paid to the biophysical basesof cell adhesion. A great deal of emphasis has been placed on treating the problem in terms of theories of colloid stability [3, 12, 231. However, in terms of colloid theory, it has proved easier to explain the absence of adhesion between cell and glass surfaces [24], cell and tooth
Cell exudation and cell adhesion
surfaces [26] and cell and virus surfaces [25], than adhesion. It has been suggestedthat one mechanism whereby a cell could overcome the potential energy barrier preventing its adhesion to a surface, would be to extrude a physicochemitally acceptable “glue” [26]. This suggestion has focused our attention on the possible relationship between extruded materials and cell adhesion. In the present experiments, the release of 51Cr from labelled cells is used as an index of cell exudation. The counts obtained from the supernatant fraction were expressed as a percentage of those in the sample of the cell suspension removed from the cultures. When the percentages were calculated on the basis of total radioactivity which includes not only counts on the supernatant medium and centrifuged cells, but also the coverslip, a similar pattern of increase was also observed. However, numerical data involving total activity were somewhat unsatisfactory when comparing and pooling data from different experiments, because removal of coverslips from the assembledculture chambers involved loss of material and, in addition, cells and other radioactive material adhered to the paraffin or silicone grease. At the commencement of the adhesion experiments, 0.3 ml of suspension containing 3 X lo5 cells/ml was placed into culture vessels of 1.3 cm internal diameter. The expected cell density on the coverslips if 100% of the cells adhered to them was 1 356 per 2 mm2 field. In typical experiments in which EAT cells sedimented on to glass, cell densities of 86 and 150 per field were observed after 5 and 90 min, respectively. The differences between 51Cr-releaseof 7.3 % after 5 min and 47.5 % after 90 min were associated with the adhesion to glass of 6.3 and 11.0% of the total cell population. The increased percentage release of 51Cr was not therefore
207
mainly due to the loss of cells in the centrifuged deposit due to their adherence to glass. It has been shown that 51Cr-label is relatively stable inside cells [2, 4, 16, 281, and that in excess of 90 % is bound to protein [17, 201. Unpublished work (Maslow, 1972) has confirmed the similarity of the labelling patterns and release from cells, of 51Crand 14C amino-acids; the techniques used by us have been described in detail elsewhere [24]. In the present experiments, the release of 51Cr is not explicable in terms of cell death and cytolysis, since both the viability and numbers of cells in individual cultures remained virtually constant over the periods of observation. The results summarized in figs l-4 show that the release of labelled material from cells closely parallels the numbers adhering to both cellular and glass substrates. When cell suspensions in glass centrifuge tubes were gently agitated to prevent them from establishing adhesions with the glass and/or each other, the release of 51Cr-labelled material was approx. 20 Y0after 90 min, compared with over 70 % in cultures containing adhering cells. Therefore, the 51Cr-releaseappears to be the result of an interaction between the cells and the surface to which they adhere, as distinct from an interaction between the cells and their suspending medium. While the experimental data indicate an obvious correlation between cell adhesion and the release of labelled material, they do not necessarily establish a causal relationship between the two. The temporal relationship between release and adhesion is also unclear, in that the material could have been released just before or just after the adhesions were formed. It has been repeatedly suggested that the nature of the adhesions between cells of different types is different in some way. For example Steinberg [21] has expressed these Exptl Cell Res 71
208
D. E. Maslow & L. Weiss
differences in terms of different adhesive strengths. The role of cellular exudates in promoting specific adhesions between cells has been described by Moscona [ll] and Humphreys [S] in the case of sponge cells, and by Lilien [7] and Rosenberg et al. [19] in the case of embryonic chicken cells. It was therefore of interest to determine whether adhesion to different (fibroblast and glass) surfaces was associated with the release of different amounts of isotopically labelled materials. Although the actual percentage release varied between experiments, no consistent differences in the average release of 51Cr per adherent cell were detected when fibroblasts adhered to glass or to fibroblast monolayers. However, a consistently higher average level of release of 51Cr per adherent cell was detected when EAT cells adhered to fibroblast monolayers, compared to glass. Thus, the two cell types studied vary in the amount of material which they release in association with their adherence to different substrates. It is of interest, in view of their different isotope-release patterns, that the EAT were maintained in suspension culture and simply transferred to the ‘adhesion’ chamber, whereas the fibroblasts were maintained in monolayer cultures, and used after trypsin dissociation. It is well known that trypsin treatment modifies the cell periphery [l, 6, 8, 13, 15, 221,and it seemspossible that such modifications could obliterate differences between the interactions of cells with different surfaces. It should however be emphasized that measurementsof percentage activity released do not reflect qualitative differences in the released material between the two situations. The present experiments were designed to study the initial stagesof the adhesion process in contrast to the work described earlier [14, 18, 271which dealt with longer experiments, Exptl
Cell Res 71
and there is therefore no disagreement between our own results and the earlier work. The authors with to thank Barbara McLaughlin and David Waite for their technical assistance and David Graham for preparing the autoradioaraphs. This work was partially supposed‘ by a June Lambert Memorial Grant from the American Cancer Society (P403-D).
REFERENCES 1. Barnard, P J, Weiss, L & Ratcliffe, T, Exptl cell res 54 (1969) 293. 2. Bunting, W L, Kiely, J M & Owen, C A, Proc sot exptl biol med 113 (1963) 370. 3. Curtis, A S G, The cell surface: its molecular role in morphogenesis. Logos Press, London (1967). Goodman, H S, Nature 190 (1961) 269. Humphreys, T, Develop biol 8 (1963) 27. Kemp, R B, Cytobios 2 (1969) 187. Lilien, J E, Develop biol 17 (1968) 657. Maslow, D E, Exptl cell res 61 (1970) 266. Moore, G E, Gemer, R E & Franklin, H A, J Am med assoc 199 (1967) 519. 10. Moore, G E, Sandberg, A A & Ulrich, K, J natl cant inst 36 (1966) 405. 11. Moscona, A A, Proc natl acad sci US 40 (1963) 142. 12. Pethica, B A, Exptl cell res, suppl. 8 (1961) 123. 13. Poste, G, Exptl cell res 65 (1971) 359. 14. Poste, G & Greenham, L W, Cytobios 2 (1970) 243. 15. Rinaldini, L M, Exptl cell res 16 (1959) 477. 16. Roaentine. G N, Jr & Placenik, B A, Transplantations 5 (1967) 1323. 17. Ronai, P M, Blood 33 (1969) 408. Rosenberg, M D, Biophys j 1 (1960) 137. ii: Rosenberg, M D, Aufderheide, K & Christianson, J, Exptl cell res 57 (1969) 449. 20. Scaife, J F & Vittorio, P V, Canad j biochem 42 (1964) 503. 21. Steinberg, M S, J exptl zoo1 173 (1970) 395. 22. Weiss, L, Exptl cell res 14 (1958) 80. 23. Weiss, L, The cell periphery, metastasis and other contact phenomena. North-Holland, Amsterdam (1967). 24. Weiss, L, Exptl cell res 51 (1968) 609. 25. Weiss, L & Horoszewicz, J S, Int j cancer 7 (1971) 149. 26. W$iss, L & Neiders, M, J periodont res 6 (1971) 27. Weiss, P, J exptl zoo1 100 (1945) 353. 28. Wigzell, H, Transplantation 3 (1965) 423.
Received June 15, 1971 Revised version received September 13, 1971
CELL EXUDATION
AND CELL ADHESION
D. E. MASLOW and L. WEISS Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, N. Y. 14203, USA
SUMMARY Experiments are described in which the exudation of 51Cr-labelled material from fibroblasts and Ehrlich ascites tumor (EAT) cells was correlated with their adhesion to cellular and noncellular surfaces, in vitro. A correlation coefficient of greater than + 0.90 was obtained for adhesion of cells to glass and the release associated with it for both cell types. The release per cell for EAT cells adhering to a fibroblast monolayer was greater than for a glass substrate. Fibroblasts showed no significant difference between adherence to glass or to a fibroblast monolayer. The apparent “substratum specificity” of Vr-release exhibited by the EAT cells, and its apparent loss by the fibroblasts is discussed.
In spite of intensive investigations, the detailed mechanisms of cell adhesion remain obscure. Although cellular exudation has been studied as one of the consequences of cell adhesion [14, 18, 271 its role in promoting cell adhesion has received comparatively little attention. In this communication we describe experiments in which the exudation of 51Cr-labelled material from cells was correlated with their adhesion to cellular and non-cellular surfaces, in vitro. MATERIAL
AND METHODS
Cell culture All cell culture medium and reagents, unless otherwise indicated, were obtained from Microbiological Associates (Bethesda, Md). Cells: (a) Fibroblasts-mouse fibroblasts of the L-929 line are maintained routinely in this laboratory on glass at 37”C, in RPM1 1640 medium [9] supplemented with 20% fetal bovine serum, in an atmosphere of 5 % CO2 in air. They were freed from glass by incubation in 0.2 % trypsin in solution, for 3 min at 37°C. (b) Ehrlich ascites tumor cells-EAT cells of the Exptl Cell Res 71
Lettre hyperdiploid strain are maintained in suspension culture in RPM1 1630 medium [lo] supplemented with 5 % calf serum. Culture vessels: Culture vessels were made by sticking Teflon rings of 1.3 cm inside diameter and 0.3 cm height, to- cleaned glass slides or coverslips, with either silicone grease or paraffin. Monolayers were obtained by adding 0.25 ml of cell suspension at a concentration of approx. 600000 cells/ml, to the chambers and incubating at 37°C for up to 24 h in a 5 % CO,-in air, high humidity atmosphere. Suspensions of labelled cells were added at concentrations of 300000 cells/ml to vessels, with or without monolayers. Labelling: Tr: Na 51CrOa, equivalent to 25 ,uCi activity, was added to 10 ml of cell suspension containing’approx. 10’ cells. After 30 min incubation at 37°C. the cells were washed with cold medium and kept at 4°C for 4 h. They were used after 2 additional washes in medium. At intervals after the addition of the cells to the chambers, the medium was removed and the suspended cells spun down in a Coleman microcentrifuge for 1 min. The supematant was removed and counted, as was the cell pellet and the coverslip, in a Packard Auto-Gamma counter. 8H: Ten /Xi of *H-thymidine was added to cultures of fibroblasts 6 h after seeding, and at 24 and 48 h thereafter. After an additional 24 h. the cells were resuspended and washed 3 times in ‘medium before use. EAT cells were incubated for 1 h with 5 &i of 8H-uridine in 5 ml of medium containing approx. 20 x 10’ cells, and used after 3 washes.
Cell exudation and cell adhesion
205
-6
in fig. 1. The correlation coefficient for the data on cell adherence and release of radioactive chromium for the typical experiment shown is +0.933. The percentage release of 51Cr in a gently agitated suspension of EAT cells after 90 min was 12.6%.
-3
Adherence of jibroblzts
-9
Fig. 1. Abscissa: time (min); ordinate: (left) O-0, % released 61Cr in the sunematant fraction: (right) O-O, average number of cells/field. EAT cell adherence and 51Cr release after settling onto a glass substrate in a typical experiment. Autoradiography: Formalin-fixed preparations were stained with Mayer hematoxylin, coated with Kodak Nuclear Track Emulsion-NTB 3, and developed after 2 weeks. Counts of adherent cells: After gently rinsing the coverslips free of non-adherent cells, with serum-free medium, the adherent cells were counted. Routinely, the cells adhering to glass were counted bv nhasecontrast microsc
to glass
The number of fibroblasts adherent to glass, increased with time during the 100 min duration of the experiments (fig. 2). During the same time period, the percentage release of 51Crincreased up to approx. 70 and 90% in the two experiments noted in fig. 2. The correlation coefficient for these adhesion and 51Cr-releasedata was greater than +0.90 in all of the experiments. The percentages of released radioactivity in the suspensions of fibroblasts prevented from adhering were 23.8 and 28.2 respectively. Adherence of EAT cells to jibroblast monolayers
The number of EAT cells adherent to fibroblast monolayers increased up to 60 min
RESULTS Adherence of EAT cells to glass
The number of EAT cells that remained adherent to glass increased with time up to 60 min. Thereafter, the number did not change significantly (fig. 1). The release of 51Cr,as measured by the percentage of counts in the supernatant fraction of that in the final cell suspension, also increased progressively up to 60 min, after which no significant increase was noted in the cultures, as shown
Fig. 2. Abscissa: time (mm); ordinate: (left) e--a, average number of cells/field; (rinht) O-O, A-A, percentage of released %r in the sup&natant fraction: Fibroblast cell adherence and 61Cr release after settling onto a glass substrate in some typical experiments. Exptl Cell Res 71
206
D. E. Maslow & L. Weiss
in culture, but not subsequently. The percentage release of 51Cr, however, increased throughout the 90 min course of these experiments. Experiments were also made in which the percentage release of 51Cr, or the number of adherent cells, was determined for simultaneous cultures of EAT cells interacting with glass or fibroblast monolayers. In these two groups of experiments, no significant differences were found between the numbers of EAT cells adherent to the glass or fibroblasts; however, the average percentage release of 51Cr was significantly greater in the case of cells adhering to the monolayer as shown in fig. 3. Comparison of the mean percentages of 51Cr released by standard t-tests, revealed that at all times studied, 0.05 >p >O.Ol. Eighteen and 19 % of 51Cr was released from the EAT cells used in two of these groups of experiments, after 90 min in suspension culture, when adhesion was prevented by gentle agitation. The adherence of fibroblast cells to a fibroblast monolayer
The numbers of fibroblast cells adhering to a fibroblast monolayer increased throughout I 60 1
40
///A0 /
I
20
I ,’ 0
60
40
0
0
30
60
9C
Fig. 4. Abscissa: time (min); ordinate: average percentage of released Wr in the supematant fraction; (left) O-O, fibroblast monolayer substrate; (right) O-O, glass substrate. Comnarison of the release of Wr from fibroblast cells settling onto a fibroblast monolayer and glass.
the 90 min course of these experiments. The numbers of cells that adhered to the monolayer were not significantly different from those adhering to a glass surface in control cultures. The releaseof the 51Crfrom the fibroblasts, adhering to a fibroblast monolayer also increased during the 90 min observed (fig. 4). The correlation coefficient for the increase in release and increase in adherent cells was greater than +0.90 in all the experiments. The percentage release of 51Crwas not significantly different (p=O.4 to 0.9) when the fibroblasts adhered to glass or to a fibroblast monolayer (fig. 4).
1
! L
r/ 01
80-
I 30
I 60
I I 90
Fig. 3. Abscissa: time (min); ordinate: average percentage of released 61Cr in the supematant fraction; (left) O-O, fibroblast monolayer substrate; (right) o-o, glass substrate. Comparison of the release of Wr from EAT cells settling onto a fibroblast monolayer and glass. Exptl Cell Res 71
DISCUSSION In recent years, much attention has been paid to the biophysical basesof cell adhesion. A great deal of emphasis has been placed on treating the problem in terms of theories of colloid stability [3, 12, 231. However, in terms of colloid theory, it has proved easier to explain the absence of adhesion between cell and glass surfaces [24], cell and tooth
Cell exudation and cell adhesion
surfaces [26] and cell and virus surfaces [25], than adhesion. It has been suggestedthat one mechanism whereby a cell could overcome the potential energy barrier preventing its adhesion to a surface, would be to extrude a physicochemitally acceptable “glue” [26]. This suggestion has focused our attention on the possible relationship between extruded materials and cell adhesion. In the present experiments, the release of 51Cr from labelled cells is used as an index of cell exudation. The counts obtained from the supernatant fraction were expressed as a percentage of those in the sample of the cell suspension removed from the cultures. When the percentages were calculated on the basis of total radioactivity which includes not only counts on the supernatant medium and centrifuged cells, but also the coverslip, a similar pattern of increase was also observed. However, numerical data involving total activity were somewhat unsatisfactory when comparing and pooling data from different experiments, because removal of coverslips from the assembledculture chambers involved loss of material and, in addition, cells and other radioactive material adhered to the paraffin or silicone grease. At the commencement of the adhesion experiments, 0.3 ml of suspension containing 3 X lo5 cells/ml was placed into culture vessels of 1.3 cm internal diameter. The expected cell density on the coverslips if 100% of the cells adhered to them was 1 356 per 2 mm2 field. In typical experiments in which EAT cells sedimented on to glass, cell densities of 86 and 150 per field were observed after 5 and 90 min, respectively. The differences between 51Cr-releaseof 7.3 % after 5 min and 47.5 % after 90 min were associated with the adhesion to glass of 6.3 and 11.0% of the total cell population. The increased percentage release of 51Cr was not therefore
207
mainly due to the loss of cells in the centrifuged deposit due to their adherence to glass. It has been shown that 51Cr-label is relatively stable inside cells [2, 4, 16, 281, and that in excess of 90 % is bound to protein [17, 201. Unpublished work (Maslow, 1972) has confirmed the similarity of the labelling patterns and release from cells, of 51Crand 14C amino-acids; the techniques used by us have been described in detail elsewhere [24]. In the present experiments, the release of 51Cr is not explicable in terms of cell death and cytolysis, since both the viability and numbers of cells in individual cultures remained virtually constant over the periods of observation. The results summarized in figs l-4 show that the release of labelled material from cells closely parallels the numbers adhering to both cellular and glass substrates. When cell suspensions in glass centrifuge tubes were gently agitated to prevent them from establishing adhesions with the glass and/or each other, the release of 51Cr-labelled material was approx. 20 Y0after 90 min, compared with over 70 % in cultures containing adhering cells. Therefore, the 51Cr-releaseappears to be the result of an interaction between the cells and the surface to which they adhere, as distinct from an interaction between the cells and their suspending medium. While the experimental data indicate an obvious correlation between cell adhesion and the release of labelled material, they do not necessarily establish a causal relationship between the two. The temporal relationship between release and adhesion is also unclear, in that the material could have been released just before or just after the adhesions were formed. It has been repeatedly suggested that the nature of the adhesions between cells of different types is different in some way. For example Steinberg [21] has expressed these Exptl Cell Res 71
208
D. E. Maslow & L. Weiss
differences in terms of different adhesive strengths. The role of cellular exudates in promoting specific adhesions between cells has been described by Moscona [ll] and Humphreys [S] in the case of sponge cells, and by Lilien [7] and Rosenberg et al. [19] in the case of embryonic chicken cells. It was therefore of interest to determine whether adhesion to different (fibroblast and glass) surfaces was associated with the release of different amounts of isotopically labelled materials. Although the actual percentage release varied between experiments, no consistent differences in the average release of 51Cr per adherent cell were detected when fibroblasts adhered to glass or to fibroblast monolayers. However, a consistently higher average level of release of 51Cr per adherent cell was detected when EAT cells adhered to fibroblast monolayers, compared to glass. Thus, the two cell types studied vary in the amount of material which they release in association with their adherence to different substrates. It is of interest, in view of their different isotope-release patterns, that the EAT were maintained in suspension culture and simply transferred to the ‘adhesion’ chamber, whereas the fibroblasts were maintained in monolayer cultures, and used after trypsin dissociation. It is well known that trypsin treatment modifies the cell periphery [l, 6, 8, 13, 15, 221,and it seemspossible that such modifications could obliterate differences between the interactions of cells with different surfaces. It should however be emphasized that measurementsof percentage activity released do not reflect qualitative differences in the released material between the two situations. The present experiments were designed to study the initial stagesof the adhesion process in contrast to the work described earlier [14, 18, 271which dealt with longer experiments, Exptl
Cell Res 71
and there is therefore no disagreement between our own results and the earlier work. The authors with to thank Barbara McLaughlin and David Waite for their technical assistance and David Graham for preparing the autoradioaraphs. This work was partially supposed‘ by a June Lambert Memorial Grant from the American Cancer Society (P403-D).
REFERENCES 1. Barnard, P J, Weiss, L & Ratcliffe, T, Exptl cell res 54 (1969) 293. 2. Bunting, W L, Kiely, J M & Owen, C A, Proc sot exptl biol med 113 (1963) 370. 3. Curtis, A S G, The cell surface: its molecular role in morphogenesis. Logos Press, London (1967). Goodman, H S, Nature 190 (1961) 269. Humphreys, T, Develop biol 8 (1963) 27. Kemp, R B, Cytobios 2 (1969) 187. Lilien, J E, Develop biol 17 (1968) 657. Maslow, D E, Exptl cell res 61 (1970) 266. Moore, G E, Gemer, R E & Franklin, H A, J Am med assoc 199 (1967) 519. 10. Moore, G E, Sandberg, A A & Ulrich, K, J natl cant inst 36 (1966) 405. 11. Moscona, A A, Proc natl acad sci US 40 (1963) 142. 12. Pethica, B A, Exptl cell res, suppl. 8 (1961) 123. 13. Poste, G, Exptl cell res 65 (1971) 359. 14. Poste, G & Greenham, L W, Cytobios 2 (1970) 243. 15. Rinaldini, L M, Exptl cell res 16 (1959) 477. 16. Roaentine. G N, Jr & Placenik, B A, Transplantations 5 (1967) 1323. 17. Ronai, P M, Blood 33 (1969) 408. Rosenberg, M D, Biophys j 1 (1960) 137. ii: Rosenberg, M D, Aufderheide, K & Christianson, J, Exptl cell res 57 (1969) 449. 20. Scaife, J F & Vittorio, P V, Canad j biochem 42 (1964) 503. 21. Steinberg, M S, J exptl zoo1 173 (1970) 395. 22. Weiss, L, Exptl cell res 14 (1958) 80. 23. Weiss, L, The cell periphery, metastasis and other contact phenomena. North-Holland, Amsterdam (1967). 24. Weiss, L, Exptl cell res 51 (1968) 609. 25. Weiss, L & Horoszewicz, J S, Int j cancer 7 (1971) 149. 26. W$iss, L & Neiders, M, J periodont res 6 (1971) 27. Weiss, P, J exptl zoo1 100 (1945) 353. 28. Wigzell, H, Transplantation 3 (1965) 423.
Received June 15, 1971 Revised version received September 13, 1971