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Cyclic variations in the ruthenium red stained coat of cells from a synchronized human lymphoblastoid line
Preliminary notes differentiation of the heterochromatin with acetic-orcein at least in some plant species, there is the chemical aspect. Although the exact chemical basis of the staining reaction of Giemsa is not well understood, it has been suggested that the composition of the stain itself is paramount in this respect 173. The same staining results obtained with orcein, an amphoteric stain which in its basic state as a strong acid solution is widely used as chromosome stain and is considered DNAspecific, underline the complexity of the situation.
465
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60.
4-o
References 1. Caspersson, T, Zech, L, Modest, E J, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128. 2. Arrighi, FE & Hsu, T C, Cytogenetics 10 (1971) 81. 3. Pardue, M L & Gall, J G, Science 168 (1970) 1356. 4. Vosa, C G & Marchi, P, Nature new biol 237 (1972) 191.
5. - Giorn bot Ital 106 (1972) 151. 6. Vosa, C G, Chromosoma 30, (1970) 366. 7. Bobrow, M, Madan, K & Pearson, P L, Nature new biol 238 (1972) 122. 0
Received January 11, 1973
Cyclic variations in the ruthenium red stained coat of cells from a synchronized human lymphoblastoid line C. ROSENFELD, M. PAINTRAND, C. CHOQUET and A. M. VENUAT, Institut de Cance’rologie et d’Immunoge’n&ique, INSERM et Association ClaudeBernard, H6pital Paul-Browse, 94 800 Villejuif, France Summary. A human lymphoblastoid cell line derived from a normal donor has been synchronized with a double thymidine block. The affinity of the cell membrane for ruthenium red was examined durina different phases of the cell cycle. The thickness of thi cell coat increased from Gl to G2. The sianificance of this finding is discussed.
Most mammalian cell surfaces are coated with a layer which can be seen histochemically by several techniques [5, 6, 81. When cells are stained with ruthenium red, according to the method of Luft [4], acid mucopolysaccharides
5
IO
15
20
25
30
Fig. 1. Abscissa: hours: ordinate: (left) i _ , % labeled ceils;(right) % mitosis. 3H-TdR labelling index and mitotic index during cell synchronization.
(AMPS) can be visualized at the ultrastructural level [2]. The thickness of this ruthenium red coloured coat (RRCC) can be measured on cells from permanent lymphoblastoid lines initiated from leukemic or non-leukemic human peripheral blood [7]. Variations in the thickness of this coat have been found on cells from the same line. Vorbrodt & Koprowski [I l] found the thickness of the RRCC to be the same for normal and transformed monolayer cell lines whereas Morgan [6] and Martinez-Palomo et al. [5] found differences in the thickness of this coat under similar conditions. More recently the effect of certain nucleotides in the culture medium on the ruthenium red affinity of the glycocalix of hamster cells transformed by oncogenic Exptl Cell Res 79 (1973)
466
Preliminary notes
Fig. 2. Electron micrograph of LHN,,
cells demonstrating the RRCC at phases of the cell cycle. j 100 000.
viruses has been described [IO]. We thought it of interest, therefore, to look for RRCC thickness changes during the cell cycle in a synchronized human lymphoblastoid cell line. Material and Methods Cells from LHN,, line initiated from an apparently normal donor were cultivated following a procedure previously described [9]. Cells were passaged twice weekly in RPM1 1640 medium supplemented with 20 % fetal calf serum, 100 U penicillin and 50 ,ug/ml streptomycin. Cells were synchronized by repeated blockage with excess thymidine [I]. Thymidine was added to the cell culture to give a final concentration Expfl Ceil Res 79 (1973)
of 2.5 mM. After incubation for 24 h, the treated cells were washed twice with prewarmed medium and resuspended in fresh medium. After a I5 h incubation a second thymidine block was performed. The cells were washed twice 24 h later and seeded into normal medium. The progress of the cell generation cycle in synchronized cultures was determined on aliquots of the cultures harvested at various intervals. The percentage of cells synthesizing DNA was determined on autoradiographs following an in vitro pulse labelling with tritiated thymidine c3H-TdR: TMM 79 B 5 Ci/mM from C.E.A., France). Two millilitres of the cell culture were olaced in a test tube and labelled with medium containing 1 &i/ml of 3HTdR and incubated for I5 min at 37°C. Four smears were fixed for 10 min in methyl alcohol and processed for autoradiography by dipping into an llford K, emulsion. 1 00&2000 cells were counted after 7 days
Preliminary notes 461 of exposure and the percentage of labelled cells determined. The technique for visualization of surface muconolvsaccharides has been nreviouslv described 171. Measurement of thickness- of the _glycocalix: kil measurements were made at a magnification of 100000 and 15 to 25 different cells were used for each set of measurements. Cuts were made perpendicularly across the cell surface and measurements made from the well defined inner leaflet to the outer surface at 1 cm intervals. The measurements are depicted on histograms with the thickness plotted on the abscissa and the frequency of occurrence of each thickness plotted on the ordinate.
Results and Discussion
After the release of the thymidine block, 65 “/ of the cells are in the S phase of the cell cycle (fig. 1). The mitotic index is at its maximum 13 h after the release of the block and allows the separation of G2 from G 1. Fig. 2 shows the glycocalix during S (3 h after release), G 2 (9 h after release), and G 1 (18.5 h). The corresponding histograms are shown in fig. 3. In S phase one can see two peaks, one at 20-25 nm and the other at 30-35 nm. In G 2 the 20-25 nm peak has disappeared and the distribution of thickness measurements has increased to beyond 30 nm. In G 1 one peak is seen and it is between 20-25 nm and the majority of the measurements are below 30 nm. Our results show a correlation between changes in cell membrane thickness and the phase of the cell cycle. The distribution of measurements in G 1 is clearly different from that in G2. In S the distribution seemsto be intermediate between Gl and G2. In fig. 1 we have shown that 35% of the cells are not in S phase. It may be that cells in G 1 are contributing to the 20-25 nm peak seenin fig. 3. These data indicate that the glycocalix thickens during S phase and even more so during G2. Ruthenium red binding is considered to be selective for acid mucopolysaccharides of the cell surface [2]. Our results suggest a relationship between DNA synthesis and an increase of surface AMPS. We cannot deter-
50 40 30 20 IO
b
I.-L IO 50 40
30
50
70
30
50
70
c
30 20 IO k IO
Fig. 3. Abscissa: membrane thickness (nm); ordinate: freauencv. (a) S: cbj G2: cc) G 1. drequen~y’ d&tribution~ histograms depicting the ruthenium red alvcocslix thickness (RRCC) at different phases of th>cell cycle. Each histogram’represents from 450 to 600 different measurements.
mine at this moment whether the ultrastructural changes of the membrane reflect synthesis of some glycoproteins on the cell surface as shown by Hayden et al. [3] in phytohemagglutinin-stimulated lymphocytes or just conformational changes of the membrane giving a higher number of exposed reactive sites without de novo synthesis. However, the cyclic dependency of the RRCC thickness and the thickening during the S-G2 phases could explain the variability of the results obtained in asynchronic cell cultures. When experiments comparing RRCC surface thickness of normal and transformed lines are reported they should be evaluated in terms of the percentage of cells in each phase of the cell cycle. Exptl Cell Res 79 (1973)
468
Preliminary notes
We are extremely grateful to Mme M. Synguelakis for her valuable technical assistance and to Dr A. Viola. This work was carried out with the aid of INSERM, contract no. 71.50061, and of DGRST, contract no: 72.7.0299.
References 1. Bootsma, D, Burdke, L & Vis, 0, Exptl cell res 33 (1964) 301. 2. Gustafson, G T & Pihl, E, Acta path microbial Stand 69 (1967) 393. 3. Havden. G A. Crowlev. G H & Jamieson, G A. J biol &em 245 (1970) 5827. 4. Luft, J H, Fed proc 25 (1966) 1773. 5. Martinez-Palomo, C, Braislovsky, C & Bernhard, W. Cancer res 29 (1969) 925. 6. Morgan, H R, J viral 2 (1968) 1133. 7. Paintrand, M & Rosenfeld, C, Compt rend acad sci 274 (1972) 415. 8. Parsons, D F & Subjek, J R, Biochim biophys acta 265 (1972) 85. 9. Rosenfeld, C, Macieira-Coelho, A, Venuat, A M, Jasmin, C & Tuan, T Q, J natl cancer inst 43 (1969) 581. 10. Torpier, G & Montagnier, L, Int j cancer 6 (1970) 529. 11. Vorbrodt, A & Koprowski, H, J natl cancer inst 43 (1969) 1241. Received January 11, 1973
The movement of single cells within solid tissue masses L. L. WISEMAN and M. S. STEINBERG, Department of Biology, The College of William and Mary, Williamsburg, Vu 23185, and Department of Biology, Princeton University, Princeton, N. J. 08540, USA Summary. Individual heart and liver cells isolated from chick embrvos labelled in ovo with SH-thvmidine were seeded, in- culture, onto the surfaces- of unlabelled, embryonic heart and liver tissue masses(both tissue fragments and cellular reaggregates). Single, labelled cells, as observed in autoradiographs, infiltrated the interiors of the tissue massesin most cases. These results might be unexpected in light of previous experiments and current notions of ‘contact inhibition of cell movement’.
When a normal fibroblast advancing on a solid culture surface collides with another fibroblast, the two cells rarely become extensively superimposed. Instead, overlapping tends to be discouraged and the colliding 1 Address reprint requests to Dr Wiseman. Exptl Cell Res 79 (1973)
cell’s motion in the direction of the collision is usually inhibited. Populations of cells displaying this behavior tend to arrange themselves as monolayers, and were originally described as showing ‘contact inhibition’ of locomotion [I, 21. It was assumed that the surrounded cells in such a monolayer were all ‘contact inhibiting’ each other’s locomotion and must consequently be immobilized [3]. Extending the concept of immobilization by encirclement from two dimensions to three, Weston & Abercrombie [4] suggested that cells in solid tissues, contacted on every side by other cells, should likewise be immobilized. To test this hypothesis, they paired labelled and unlabelled bits of chick embryonic heart tissue and of chick embryonic liver tissue, allowed these homonomic pairs to fuse and round up, and later examined the interface autoradiographically for signs of intermixing, little of which was observed. They interpreted their observations as supporting the hypothesis that locomotion of individual cells in these solid tissue massesis ‘contact-inhibited’. Yet circumstances exist in which such cells can translocate within 3-dimensional tissue masses. For example, when populations of dispersed cells from two different embryonic organs are intermixed and brought together in culture, sorting-out typically occurs. One population tends to take up an internal and the other an external position (see [5] for review). Starting from a random mixture, the cells reorganize into two discrete ‘phases’. Such a reorganization requires cell movement over distances many times the diameter of a single cell, with the individual cells presumably moving over one another in the process. Trinkaus & Lentz [6] have observed directly the sorting-out of embryonic chick heart and pigmented retinal cells, and report that small, isolated pigmented cell clusters moved only small distances (10-20 pm), if
465
801
60.
4-o
References 1. Caspersson, T, Zech, L, Modest, E J, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128. 2. Arrighi, FE & Hsu, T C, Cytogenetics 10 (1971) 81. 3. Pardue, M L & Gall, J G, Science 168 (1970) 1356. 4. Vosa, C G & Marchi, P, Nature new biol 237 (1972) 191.
5. - Giorn bot Ital 106 (1972) 151. 6. Vosa, C G, Chromosoma 30, (1970) 366. 7. Bobrow, M, Madan, K & Pearson, P L, Nature new biol 238 (1972) 122. 0
Received January 11, 1973
Cyclic variations in the ruthenium red stained coat of cells from a synchronized human lymphoblastoid line C. ROSENFELD, M. PAINTRAND, C. CHOQUET and A. M. VENUAT, Institut de Cance’rologie et d’Immunoge’n&ique, INSERM et Association ClaudeBernard, H6pital Paul-Browse, 94 800 Villejuif, France Summary. A human lymphoblastoid cell line derived from a normal donor has been synchronized with a double thymidine block. The affinity of the cell membrane for ruthenium red was examined durina different phases of the cell cycle. The thickness of thi cell coat increased from Gl to G2. The sianificance of this finding is discussed.
Most mammalian cell surfaces are coated with a layer which can be seen histochemically by several techniques [5, 6, 81. When cells are stained with ruthenium red, according to the method of Luft [4], acid mucopolysaccharides
5
IO
15
20
25
30
Fig. 1. Abscissa: hours: ordinate: (left) i _ , % labeled ceils;(right) % mitosis. 3H-TdR labelling index and mitotic index during cell synchronization.
(AMPS) can be visualized at the ultrastructural level [2]. The thickness of this ruthenium red coloured coat (RRCC) can be measured on cells from permanent lymphoblastoid lines initiated from leukemic or non-leukemic human peripheral blood [7]. Variations in the thickness of this coat have been found on cells from the same line. Vorbrodt & Koprowski [I l] found the thickness of the RRCC to be the same for normal and transformed monolayer cell lines whereas Morgan [6] and Martinez-Palomo et al. [5] found differences in the thickness of this coat under similar conditions. More recently the effect of certain nucleotides in the culture medium on the ruthenium red affinity of the glycocalix of hamster cells transformed by oncogenic Exptl Cell Res 79 (1973)
466
Preliminary notes
Fig. 2. Electron micrograph of LHN,,
cells demonstrating the RRCC at phases of the cell cycle. j 100 000.
viruses has been described [IO]. We thought it of interest, therefore, to look for RRCC thickness changes during the cell cycle in a synchronized human lymphoblastoid cell line. Material and Methods Cells from LHN,, line initiated from an apparently normal donor were cultivated following a procedure previously described [9]. Cells were passaged twice weekly in RPM1 1640 medium supplemented with 20 % fetal calf serum, 100 U penicillin and 50 ,ug/ml streptomycin. Cells were synchronized by repeated blockage with excess thymidine [I]. Thymidine was added to the cell culture to give a final concentration Expfl Ceil Res 79 (1973)
of 2.5 mM. After incubation for 24 h, the treated cells were washed twice with prewarmed medium and resuspended in fresh medium. After a I5 h incubation a second thymidine block was performed. The cells were washed twice 24 h later and seeded into normal medium. The progress of the cell generation cycle in synchronized cultures was determined on aliquots of the cultures harvested at various intervals. The percentage of cells synthesizing DNA was determined on autoradiographs following an in vitro pulse labelling with tritiated thymidine c3H-TdR: TMM 79 B 5 Ci/mM from C.E.A., France). Two millilitres of the cell culture were olaced in a test tube and labelled with medium containing 1 &i/ml of 3HTdR and incubated for I5 min at 37°C. Four smears were fixed for 10 min in methyl alcohol and processed for autoradiography by dipping into an llford K, emulsion. 1 00&2000 cells were counted after 7 days
Preliminary notes 461 of exposure and the percentage of labelled cells determined. The technique for visualization of surface muconolvsaccharides has been nreviouslv described 171. Measurement of thickness- of the _glycocalix: kil measurements were made at a magnification of 100000 and 15 to 25 different cells were used for each set of measurements. Cuts were made perpendicularly across the cell surface and measurements made from the well defined inner leaflet to the outer surface at 1 cm intervals. The measurements are depicted on histograms with the thickness plotted on the abscissa and the frequency of occurrence of each thickness plotted on the ordinate.
Results and Discussion
After the release of the thymidine block, 65 “/ of the cells are in the S phase of the cell cycle (fig. 1). The mitotic index is at its maximum 13 h after the release of the block and allows the separation of G2 from G 1. Fig. 2 shows the glycocalix during S (3 h after release), G 2 (9 h after release), and G 1 (18.5 h). The corresponding histograms are shown in fig. 3. In S phase one can see two peaks, one at 20-25 nm and the other at 30-35 nm. In G 2 the 20-25 nm peak has disappeared and the distribution of thickness measurements has increased to beyond 30 nm. In G 1 one peak is seen and it is between 20-25 nm and the majority of the measurements are below 30 nm. Our results show a correlation between changes in cell membrane thickness and the phase of the cell cycle. The distribution of measurements in G 1 is clearly different from that in G2. In S the distribution seemsto be intermediate between Gl and G2. In fig. 1 we have shown that 35% of the cells are not in S phase. It may be that cells in G 1 are contributing to the 20-25 nm peak seenin fig. 3. These data indicate that the glycocalix thickens during S phase and even more so during G2. Ruthenium red binding is considered to be selective for acid mucopolysaccharides of the cell surface [2]. Our results suggest a relationship between DNA synthesis and an increase of surface AMPS. We cannot deter-
50 40 30 20 IO
b
I.-L IO 50 40
30
50
70
30
50
70
c
30 20 IO k IO
Fig. 3. Abscissa: membrane thickness (nm); ordinate: freauencv. (a) S: cbj G2: cc) G 1. drequen~y’ d&tribution~ histograms depicting the ruthenium red alvcocslix thickness (RRCC) at different phases of th>cell cycle. Each histogram’represents from 450 to 600 different measurements.
mine at this moment whether the ultrastructural changes of the membrane reflect synthesis of some glycoproteins on the cell surface as shown by Hayden et al. [3] in phytohemagglutinin-stimulated lymphocytes or just conformational changes of the membrane giving a higher number of exposed reactive sites without de novo synthesis. However, the cyclic dependency of the RRCC thickness and the thickening during the S-G2 phases could explain the variability of the results obtained in asynchronic cell cultures. When experiments comparing RRCC surface thickness of normal and transformed lines are reported they should be evaluated in terms of the percentage of cells in each phase of the cell cycle. Exptl Cell Res 79 (1973)
468
Preliminary notes
We are extremely grateful to Mme M. Synguelakis for her valuable technical assistance and to Dr A. Viola. This work was carried out with the aid of INSERM, contract no. 71.50061, and of DGRST, contract no: 72.7.0299.
References 1. Bootsma, D, Burdke, L & Vis, 0, Exptl cell res 33 (1964) 301. 2. Gustafson, G T & Pihl, E, Acta path microbial Stand 69 (1967) 393. 3. Havden. G A. Crowlev. G H & Jamieson, G A. J biol &em 245 (1970) 5827. 4. Luft, J H, Fed proc 25 (1966) 1773. 5. Martinez-Palomo, C, Braislovsky, C & Bernhard, W. Cancer res 29 (1969) 925. 6. Morgan, H R, J viral 2 (1968) 1133. 7. Paintrand, M & Rosenfeld, C, Compt rend acad sci 274 (1972) 415. 8. Parsons, D F & Subjek, J R, Biochim biophys acta 265 (1972) 85. 9. Rosenfeld, C, Macieira-Coelho, A, Venuat, A M, Jasmin, C & Tuan, T Q, J natl cancer inst 43 (1969) 581. 10. Torpier, G & Montagnier, L, Int j cancer 6 (1970) 529. 11. Vorbrodt, A & Koprowski, H, J natl cancer inst 43 (1969) 1241. Received January 11, 1973
The movement of single cells within solid tissue masses L. L. WISEMAN and M. S. STEINBERG, Department of Biology, The College of William and Mary, Williamsburg, Vu 23185, and Department of Biology, Princeton University, Princeton, N. J. 08540, USA Summary. Individual heart and liver cells isolated from chick embrvos labelled in ovo with SH-thvmidine were seeded, in- culture, onto the surfaces- of unlabelled, embryonic heart and liver tissue masses(both tissue fragments and cellular reaggregates). Single, labelled cells, as observed in autoradiographs, infiltrated the interiors of the tissue massesin most cases. These results might be unexpected in light of previous experiments and current notions of ‘contact inhibition of cell movement’.
When a normal fibroblast advancing on a solid culture surface collides with another fibroblast, the two cells rarely become extensively superimposed. Instead, overlapping tends to be discouraged and the colliding 1 Address reprint requests to Dr Wiseman. Exptl Cell Res 79 (1973)
cell’s motion in the direction of the collision is usually inhibited. Populations of cells displaying this behavior tend to arrange themselves as monolayers, and were originally described as showing ‘contact inhibition’ of locomotion [I, 21. It was assumed that the surrounded cells in such a monolayer were all ‘contact inhibiting’ each other’s locomotion and must consequently be immobilized [3]. Extending the concept of immobilization by encirclement from two dimensions to three, Weston & Abercrombie [4] suggested that cells in solid tissues, contacted on every side by other cells, should likewise be immobilized. To test this hypothesis, they paired labelled and unlabelled bits of chick embryonic heart tissue and of chick embryonic liver tissue, allowed these homonomic pairs to fuse and round up, and later examined the interface autoradiographically for signs of intermixing, little of which was observed. They interpreted their observations as supporting the hypothesis that locomotion of individual cells in these solid tissue massesis ‘contact-inhibited’. Yet circumstances exist in which such cells can translocate within 3-dimensional tissue masses. For example, when populations of dispersed cells from two different embryonic organs are intermixed and brought together in culture, sorting-out typically occurs. One population tends to take up an internal and the other an external position (see [5] for review). Starting from a random mixture, the cells reorganize into two discrete ‘phases’. Such a reorganization requires cell movement over distances many times the diameter of a single cell, with the individual cells presumably moving over one another in the process. Trinkaus & Lentz [6] have observed directly the sorting-out of embryonic chick heart and pigmented retinal cells, and report that small, isolated pigmented cell clusters moved only small distances (10-20 pm), if