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Sensitivity of JLSV-9 cells to Moloney leukemia virus in relation to cell cycle
230
L. Gergely et al.
ism of cilia regeneration seemsto be somewhat complex and further analyses are needed. In the present experiment, it is also observed that cells which were isolated from embryos just before the formation of the original cilia cannot form cilia even in normal sea water as long as they are kept unaggregated [l]. This fact strongly suggeststhat the effects of cell dissociation upon the formation of original cilia are also the same as those upon cilia regeneration. The author wishes to thank Professor H. Terayama and Dr K. Yamagami for their encouragement and for reading through this manuscript.
REFERENCES 1. Amemiya, S. Unpublished. 2. Auclair, W & Siegel, B W, Science 154 (1966) 913. 3. Giudice, G, Develop biol 5 (1962) 402. 4. Iwaikawa, Y, Embryologia 9 (1967) 287. 5. Pfohl. R J & Giudice. G. Biochim biophys _ _ acta 142 (1967) 263. 6. Sconzo, G, Pirrone, A M, Mutolo, V & Giudice, G, Biochim biophys acta 199 (1970) 441. 7. Yasumasu, I & Koshihara, H, Zoo1 mag 76 (1967) 263. Received August 28, 1970 Revised version received October 12, 1970
Sensitivity of JLSV-9 cells to Moloney leukemia virus in relation to cell cycle L. GERGELY, M. CIKES, E. KLEIN, E. M. FENYG and S. FRIBERG, Department of Tumour Biology, Karolinska Sweden
Instituter,
S-104 01 Stockholm
60,
The JLSV-9 mouse bone marrow line can be infected with Moloney leukemia virus (MLV). Nordenskjold et al. [3] described the dosedependent appearance of virally determined surface antigen. According to earlier studies of various authors, with partially or fully synchronized cultures, appropriate target cells are most susceptible to RSV, MSV and rat virus during the S phase of their cycle [l, Exptl Cell Res 64
Fig. 1. The presence of Moloney virus determined
surface antigen on JLSV-9 cells as seen by indirect immunofluorescence.
5-91. We have studied the sensitivity of synchronized JLSV-9 cells to MLV infection at hourly intervals during one growth cycle by the induction of virally determined membrane antigen. Material and Methods The cells were svnchronized bv Colcemid blockade (0.04 mg/ml) as described previously [4]. During the cell cvcle we followed (a) DNA svnthesis bv SHthymihine incorporation into TCA insoluble precipitate (spec. act. 18.3 Ci/mmole; pulse time 30 min; dosage 0.2 pCi/ml), (b) the change of the modal cell volume (Coulter Counter, model B), and (c) the mitotic index. After arresting 95 % of the cells in metaohase. they were washed and resusuended in prewarmed fresh MEM with 10 % fetal calf serum and seeded in milk dilution bottles (20 ml of 0.8 x lo5 cells/ml). Every hour three bottles were removed: one to measure 3H-thymidine incorporation after a 30 min pulse, one to estimate the modal cell volume and the mitotic index on a trypsinized sample, and one to infect with MLV. For the viral source a 50% homogenate of the MLV-induced YAC ascites lymphoma diluted 1: 100 in MEM was used. (On previous experiments this dose of the virus induced detectable cell surface antigen in 14 days.) The bottle with cells to be infected was incubated with 1 ml of the virus preparation at 37°C for 60 min. The unadsorbed virus was removed by washing the cells with 10 ml medium; 10 ml new medium was then added. Subsequently, the infected cultures were maintained and split 1: 10 every second day. The appearance of the virally determined surface antigen was detected by indirect membrane immunofluorescence on detached viable cells. The serum reagent for this test was obtained by immunizing. A.BY x DBA/2 Fl mice with syngeneic Moloney leukemia cells as described pre-
Cell sensitivity related to cell cycle
231
viously [3]. After exposing the target cells to a 1 :5 dilution of the anti-MLV serum, the cells were washed and stained with FITC conjugated goat anti-mouse gammaglobulin, diluted 1: 20 (Hyland Labs, Los Angeles). Care was taken to use the cultures at a comparable cell density. The membrane immunofluorescence test was performed on cells originating from complete monolayers (second day after splitting). It was read by counting the proportion of membrane-positive cells in samples of 100 cells, using the same criteria as previously described [3]. The experiment was performed on two different occasions using the same virus source. Essentially identical results were obtained. Fig. 2 shows the variations of 3H-thymidine incorporation, the mitotic index and modal cell volume during one cell cycle. The curves demonstrate successful synchronization. 50 40 30
1-1
Ot
2
/ /9 \ / ,/o 0 b “‘” /‘O >,,O’ 0 O /“Y __ , 3
I ‘: /\ j - i ‘0:
0
0
5
10
15
Fig. 2. Abscissa: time after Colcemid release (hours); ordinate: (left) 3H-TdR incorporation (cpm x 103) V---V.
Events during growth of synchronized JLSV-9 cells. Cells were arrested in metaphase by brief Colcemid treatment, resuspended in fresh prewarmed medium, and distributed in replicate milk dilution bottles (20 ml cell suspension per bottle) at a concentration of 0.8 x lo5 cells/ml. At indicated time points after Colcemid release, monolayers were trypsinized and cell volume, mitotic index, and SH-TdR incorporation were determined. At the same time points, cells growing in parallel bottles were infected with MLV as described in text.
5
IO
15
Fig. 3. Abscissa: infection performed after Colcemid release (hours); ordinate: “: immunofluorescencepositive cells. Proportion of immunofluorescence-positive cells at 18, 22 and 35 days after MLV infection of JLSV-9 cells. The abscissa indicates the infection event as related to the release of Colcemid blockade, i.e. related to the cell cycle as marked at the top of the figure.
Fig. 3 demonstrates the appearance of the membrane antigen on culture lines derived from infection at different times (hourly interval) during the cell cycle. No antigenpositive cells were observed before the cultures had been maintained 18 days. At day 18 such cells appeared. Their proportion was distinctly higher in the samples derived from infection events 2, 3, 4 or 5 h after metaphase arrest. These times correspond to the Gl and early S phase of the cell cycle. The last sample also showed a somewhat higher percentage of positive cells; this sample represents a line derived from a culture ExptI Cell Res 64
232
W. Bernhard & S. Avrameas
L.C. is reciDient of a fellowshio from the Swedish infected when a fraction of cells had already Institute, 04 leave from Instituie of Microbiology, passed the second mitotic wave and were in Medical University of Debrecen, Hungary. M.C. is recipient of a Fellowship from the Interthe following GI phase. At 22 days following national Agency for Research on Cancer. infection, the percentage of stained cells had This work was supported by the grants from the increased in all cell lines, although the lines Swedish Cancer Society, the Damon Runvon Fund for Cancer Research (DRG 1064), the Medical Retaken from Gl and early S still showed a search Council, and the Jane Coffin Childs Memorial relative higher percentage of positive cells. Fund for Medical Research. As time went on, cell lines initially containing a low number of antigen-positive cells caught REFERENCES up with the others. By day 3.5, there were no 1. Bader, J P, Subviral carcenogenesis (ed Y Ito) differences. As mentioned before, our prep. 144. Aichi Cancer Center, Nagoya (1966). ’ vious experiments indicated that the time 2. Cikes, M, Friberg, S Jr. Submitted for publication (1970). preceding the appearance of virally deter3. NordenskjGld, B A, Klein, E, Tachibana, T & mined membrane antigens depended on the FenyG, E M, J natl cancer inst 44 (1970) 403. 4. Romsdahl, H M, Exptl cell res 50 (1968) 463. virus dose. Thus the delay in the appearance 5. Temin, H M, J cell physiol 69 (1967) 53. of the antigen in lines derived from cells 6. Pennant, R W, Layman, K R & Hand, R E Jr, J virol 4 (1969) 872. infected at cetain phases of the cycle indicates 7. Yoshikura, H, Hirokawa, Y, Ikawa, Y & Sugano, a difference in cellular sensitivity to the virus H, Int j cancer 3 (1968) 743. infection. The Gl and early S phase of the 8. Yoshikura, H, Exptl cell res 52 (1968) 445. 9. - J gen virol 6 (1970) 183. JLSV-9 cell cycle appear to be most sensitive. Received August 26, 1970 This observation is in line with previously reported data for other viruses [I, 5-91. The cell-cycle dependent difference in senUltrastructural visualization of cellular sitivity could be due to at least two factors: carbohydrate components by means of (1) the density of cell surface receptors, reconcanavalin A quired for virus adsorption, could be higher during the early phases of cell cycle; (2) cel- W. BERNHARD and S. AVRAMEAS, Institut de Scientifiques SW le Cancer, 94 Yillejuif, lular DNA synthesis may be required for the Recherches France early steps of virus multiplication. Membrane antigen components, such as Summary genetically detemined H-2 antigens and A plant agglutinin is used to visualize with the electron microscope carbohydrate-carrying cellular comcertain virally determined surface antigens ponents, in particular, the cell coat. Concanavalin A has two reactive groups. One binds specifically to as well, have been found to undergo a cyclic certain sugars of the cell, the other acts as receptor variation in expression during the cell cycle for the carbohydrate-containing peroxidase, the presence of which can be revealed with the diaminoben[2]. It is conceivable that virus receptors zidine reaction. required for the adsorption and/or penetration of MLV, whether they are related to There already exists a variety of methods for such membrane antigen components or not, the ultrastructural demonstration of polyundergo a similar cyclic variation in expressaccharides or the carbohydrate components sion. This could be related to the time when of proteins or lipids [2, 5, 6, 7, 8, 9, lo]. essential constituents of the cell membrane They have been used to visualize the cell are synthesized during the cell cycle, or to the coat or glycocalyx and some of them can period in which the outer membrane changes also be applied to localize intracellular sugars while the cell prepares for mitosis. [6, 7, 9, lo]. All of them are based on cytoExptl Cell Res 64
L. Gergely et al.
ism of cilia regeneration seemsto be somewhat complex and further analyses are needed. In the present experiment, it is also observed that cells which were isolated from embryos just before the formation of the original cilia cannot form cilia even in normal sea water as long as they are kept unaggregated [l]. This fact strongly suggeststhat the effects of cell dissociation upon the formation of original cilia are also the same as those upon cilia regeneration. The author wishes to thank Professor H. Terayama and Dr K. Yamagami for their encouragement and for reading through this manuscript.
REFERENCES 1. Amemiya, S. Unpublished. 2. Auclair, W & Siegel, B W, Science 154 (1966) 913. 3. Giudice, G, Develop biol 5 (1962) 402. 4. Iwaikawa, Y, Embryologia 9 (1967) 287. 5. Pfohl. R J & Giudice. G. Biochim biophys _ _ acta 142 (1967) 263. 6. Sconzo, G, Pirrone, A M, Mutolo, V & Giudice, G, Biochim biophys acta 199 (1970) 441. 7. Yasumasu, I & Koshihara, H, Zoo1 mag 76 (1967) 263. Received August 28, 1970 Revised version received October 12, 1970
Sensitivity of JLSV-9 cells to Moloney leukemia virus in relation to cell cycle L. GERGELY, M. CIKES, E. KLEIN, E. M. FENYG and S. FRIBERG, Department of Tumour Biology, Karolinska Sweden
Instituter,
S-104 01 Stockholm
60,
The JLSV-9 mouse bone marrow line can be infected with Moloney leukemia virus (MLV). Nordenskjold et al. [3] described the dosedependent appearance of virally determined surface antigen. According to earlier studies of various authors, with partially or fully synchronized cultures, appropriate target cells are most susceptible to RSV, MSV and rat virus during the S phase of their cycle [l, Exptl Cell Res 64
Fig. 1. The presence of Moloney virus determined
surface antigen on JLSV-9 cells as seen by indirect immunofluorescence.
5-91. We have studied the sensitivity of synchronized JLSV-9 cells to MLV infection at hourly intervals during one growth cycle by the induction of virally determined membrane antigen. Material and Methods The cells were svnchronized bv Colcemid blockade (0.04 mg/ml) as described previously [4]. During the cell cvcle we followed (a) DNA svnthesis bv SHthymihine incorporation into TCA insoluble precipitate (spec. act. 18.3 Ci/mmole; pulse time 30 min; dosage 0.2 pCi/ml), (b) the change of the modal cell volume (Coulter Counter, model B), and (c) the mitotic index. After arresting 95 % of the cells in metaohase. they were washed and resusuended in prewarmed fresh MEM with 10 % fetal calf serum and seeded in milk dilution bottles (20 ml of 0.8 x lo5 cells/ml). Every hour three bottles were removed: one to measure 3H-thymidine incorporation after a 30 min pulse, one to estimate the modal cell volume and the mitotic index on a trypsinized sample, and one to infect with MLV. For the viral source a 50% homogenate of the MLV-induced YAC ascites lymphoma diluted 1: 100 in MEM was used. (On previous experiments this dose of the virus induced detectable cell surface antigen in 14 days.) The bottle with cells to be infected was incubated with 1 ml of the virus preparation at 37°C for 60 min. The unadsorbed virus was removed by washing the cells with 10 ml medium; 10 ml new medium was then added. Subsequently, the infected cultures were maintained and split 1: 10 every second day. The appearance of the virally determined surface antigen was detected by indirect membrane immunofluorescence on detached viable cells. The serum reagent for this test was obtained by immunizing. A.BY x DBA/2 Fl mice with syngeneic Moloney leukemia cells as described pre-
Cell sensitivity related to cell cycle
231
viously [3]. After exposing the target cells to a 1 :5 dilution of the anti-MLV serum, the cells were washed and stained with FITC conjugated goat anti-mouse gammaglobulin, diluted 1: 20 (Hyland Labs, Los Angeles). Care was taken to use the cultures at a comparable cell density. The membrane immunofluorescence test was performed on cells originating from complete monolayers (second day after splitting). It was read by counting the proportion of membrane-positive cells in samples of 100 cells, using the same criteria as previously described [3]. The experiment was performed on two different occasions using the same virus source. Essentially identical results were obtained. Fig. 2 shows the variations of 3H-thymidine incorporation, the mitotic index and modal cell volume during one cell cycle. The curves demonstrate successful synchronization. 50 40 30
1-1
Ot
2
/ /9 \ / ,/o 0 b “‘” /‘O >,,O’ 0 O /“Y __ , 3
I ‘: /\ j - i ‘0:
0
0
5
10
15
Fig. 2. Abscissa: time after Colcemid release (hours); ordinate: (left) 3H-TdR incorporation (cpm x 103) V---V.
Events during growth of synchronized JLSV-9 cells. Cells were arrested in metaphase by brief Colcemid treatment, resuspended in fresh prewarmed medium, and distributed in replicate milk dilution bottles (20 ml cell suspension per bottle) at a concentration of 0.8 x lo5 cells/ml. At indicated time points after Colcemid release, monolayers were trypsinized and cell volume, mitotic index, and SH-TdR incorporation were determined. At the same time points, cells growing in parallel bottles were infected with MLV as described in text.
5
IO
15
Fig. 3. Abscissa: infection performed after Colcemid release (hours); ordinate: “: immunofluorescencepositive cells. Proportion of immunofluorescence-positive cells at 18, 22 and 35 days after MLV infection of JLSV-9 cells. The abscissa indicates the infection event as related to the release of Colcemid blockade, i.e. related to the cell cycle as marked at the top of the figure.
Fig. 3 demonstrates the appearance of the membrane antigen on culture lines derived from infection at different times (hourly interval) during the cell cycle. No antigenpositive cells were observed before the cultures had been maintained 18 days. At day 18 such cells appeared. Their proportion was distinctly higher in the samples derived from infection events 2, 3, 4 or 5 h after metaphase arrest. These times correspond to the Gl and early S phase of the cell cycle. The last sample also showed a somewhat higher percentage of positive cells; this sample represents a line derived from a culture ExptI Cell Res 64
232
W. Bernhard & S. Avrameas
L.C. is reciDient of a fellowshio from the Swedish infected when a fraction of cells had already Institute, 04 leave from Instituie of Microbiology, passed the second mitotic wave and were in Medical University of Debrecen, Hungary. M.C. is recipient of a Fellowship from the Interthe following GI phase. At 22 days following national Agency for Research on Cancer. infection, the percentage of stained cells had This work was supported by the grants from the increased in all cell lines, although the lines Swedish Cancer Society, the Damon Runvon Fund for Cancer Research (DRG 1064), the Medical Retaken from Gl and early S still showed a search Council, and the Jane Coffin Childs Memorial relative higher percentage of positive cells. Fund for Medical Research. As time went on, cell lines initially containing a low number of antigen-positive cells caught REFERENCES up with the others. By day 3.5, there were no 1. Bader, J P, Subviral carcenogenesis (ed Y Ito) differences. As mentioned before, our prep. 144. Aichi Cancer Center, Nagoya (1966). ’ vious experiments indicated that the time 2. Cikes, M, Friberg, S Jr. Submitted for publication (1970). preceding the appearance of virally deter3. NordenskjGld, B A, Klein, E, Tachibana, T & mined membrane antigens depended on the FenyG, E M, J natl cancer inst 44 (1970) 403. 4. Romsdahl, H M, Exptl cell res 50 (1968) 463. virus dose. Thus the delay in the appearance 5. Temin, H M, J cell physiol 69 (1967) 53. of the antigen in lines derived from cells 6. Pennant, R W, Layman, K R & Hand, R E Jr, J virol 4 (1969) 872. infected at cetain phases of the cycle indicates 7. Yoshikura, H, Hirokawa, Y, Ikawa, Y & Sugano, a difference in cellular sensitivity to the virus H, Int j cancer 3 (1968) 743. infection. The Gl and early S phase of the 8. Yoshikura, H, Exptl cell res 52 (1968) 445. 9. - J gen virol 6 (1970) 183. JLSV-9 cell cycle appear to be most sensitive. Received August 26, 1970 This observation is in line with previously reported data for other viruses [I, 5-91. The cell-cycle dependent difference in senUltrastructural visualization of cellular sitivity could be due to at least two factors: carbohydrate components by means of (1) the density of cell surface receptors, reconcanavalin A quired for virus adsorption, could be higher during the early phases of cell cycle; (2) cel- W. BERNHARD and S. AVRAMEAS, Institut de Scientifiques SW le Cancer, 94 Yillejuif, lular DNA synthesis may be required for the Recherches France early steps of virus multiplication. Membrane antigen components, such as Summary genetically detemined H-2 antigens and A plant agglutinin is used to visualize with the electron microscope carbohydrate-carrying cellular comcertain virally determined surface antigens ponents, in particular, the cell coat. Concanavalin A has two reactive groups. One binds specifically to as well, have been found to undergo a cyclic certain sugars of the cell, the other acts as receptor variation in expression during the cell cycle for the carbohydrate-containing peroxidase, the presence of which can be revealed with the diaminoben[2]. It is conceivable that virus receptors zidine reaction. required for the adsorption and/or penetration of MLV, whether they are related to There already exists a variety of methods for such membrane antigen components or not, the ultrastructural demonstration of polyundergo a similar cyclic variation in expressaccharides or the carbohydrate components sion. This could be related to the time when of proteins or lipids [2, 5, 6, 7, 8, 9, lo]. essential constituents of the cell membrane They have been used to visualize the cell are synthesized during the cell cycle, or to the coat or glycocalyx and some of them can period in which the outer membrane changes also be applied to localize intracellular sugars while the cell prepares for mitosis. [6, 7, 9, lo]. All of them are based on cytoExptl Cell Res 64