Planta (1986)168:101-105
P l a n t a 9 Springer-Verlag 1986
Cell-cycle arrest of plant suspension cultures by tunicamycin C. Ettlinger, J. Schindler and L. Lehle Fakult/it ffir Biologie und Vorklinische Medizin, Universit/it Regensburg, Universitfitsstrasse 31, D-8400 Regensburg, Federal Republic of Germany
Abstract. The effect of tunicamycin, an inhibitor
of N-glycosylation of proteins, on growth and on synthesis of D N A and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1-1 gg. ml-1 tunicamycin, cell division and D N A synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear D N A content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the GI phase, as was recently postulated for animal and yeast cells. Key words: Catharanthus (cell cycle) - Cell cycle - Glycoprotein - Nicotiana (cell cycle) - Tunicamycin.
Introduction
Our understanding of the biochemical events of the eucaryotic cell and division cycle is far from complete. Although the pioneering experiments leading to the concept of four defined phases, G1, S, G2 and M, of the division cycle were made on a plant system (root tips of Vicia faba, Howard and Pelc 1951, 1953), cell cultures of higher plants have received less attention than animal cells (for reviews' see Gould 1984; Bryant 1976). There is evidence that the G1 and G2 phases serve as principal control points. In many merAbbreviations: TCA = trichloroacetic acid; TM = tunicamycin
istems, carbohydrate starvation leaves cells in GI or G2 (Van't H o f 1973). Deprivation of auxin from carrot cell culture leads to arrest in G1 (Nishi et al. 1977), and also cytokinin, gibberellin or abscisic acid seem to have control functions (for review, see Evans 1983; Nagl 1972). With root meristems' it was demonstrated that initiation of cell differentiation occurs in either GI or G2 (Roberts 1976). Further, the rate-limiting effect of temperature on growth in suspension cultures would also appear to be exerted during G1 (Gould 1977). Studies in animal cells (Pardee 1974; Campisi et al. 1982) and yeast (Hartwell 1974) have shown that the major regulatory events leading to proliferation in these organisms occur within a limited period during the G1 phase: cells beyond the socalled "restriction point" or " s t a r t " become committed to another cell cycle. More recently, investigations with cell lines from yeast (Arnold and Tanner 1982), Burkitt lymphoma (Nishikawa et al. 1980) and rat hepatoma (Savage and Baur 1983) have indicated that protein glycosylation is needed for transition from G1 to S. The present report extends this observation to higher-plant cells, and suggests that the phenomenon might have general relevance for all eucaryotic cells. It will be shown that tunicamycin, an inhibitor of N-glycosylation, arrests suspension cultures from Catharanthus at phase GI.
Materials and methods Material. Cells were grown in liquid suspension culture: Nicotiana tabacum in LS-medium (Linsmaier and Skoog 1965)
supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2 mg -1-1) and kinetin (0.03 mg-1 - 1); Catharanthus rosea in MSmedium (Murashige and Skoog 1962) in the presence of 2,4-D (0.44 mg ml 2). Cells were cultivated in the dark on a gyratory shaker at 110 rpm and 29 ~ C and subcultivated every week.
102
C. Ettlinger et al. : Cell-cycle arrest by tunicamycin
Methods of cell-cycle analysis. The D N A content was determined essentially according to Adams (1980) from 5-ml culture aliquots which were washed twice with 5 ml 0.85% NaC1 and once with 5% trichloroacetic acid (TCA). Calf thymus D N A was used as standard. Protein from cells was determined following the procedure of Lowry et al. (1951) after washing cells with 0.85% NaC1 and extracting it with 2 N N a O H at 85~ for 100 min. Aliquots of the clear supernatant were diluted with water and assayed. Protein content of the medium was determined after removal of cells by centrifugation from a measured aliquot. Nuclear D N A content of Feulgen-stained squashes was measured with a Zeiss scanning cytophotometer 05 (Zeiss, Oberkochem, F R G ) connected via CAMAC-crate to a Dietz 621 x 2 minicomputer (Dietz, FRG) as described by de Vries and Schindler (1984). Feulgen staining was carried out with p-rosanilin (Merck, Darmstadt, FRG) after strictly controlled fixation and acid hydrolysis. The cell culture (1 ml) was fixed twice for 20 min with 5 ml ethanol: acetic acid (3 : 1 ; v/v), applied to a slide and squashed. After hydrolysis in 1 N HC1 for 10 rain at 60 ~ C the specimens were stained for 3 h. Absorption of metaphase or telophase nuclei was equated with D N A content of G2-phase cells. To obtain the mitotic index (percentage number of mitotic figures divided by total nuclei counted) about 1500 cells were evaluated from the same preparation.
once in fresh medium without glucose and resuspended in the same medium. Tunicamycin (TM) was purchased from Calbiochem (La Jolla, Cal., USA) and was added as a filter-sterilized, aqueous solution after dissolving it in a small amount of 0.1 N N a O H and subsequent adjustment to pH 7.2. Controls contained the same aliquot of liquid without TM.
Results
Effects of tunicamycin on growth, and on synthesis o f D N A and protein. Tunicamycin has been shown to inhibit N-glycosylation of proteins by preventing the first reaction of the dolichol pathway, the transfer of N-acetylglucosamine 1-phosphate to the lipid intermediate carrier (Lehle and Tanner 1976; Takatsuki et al. 1975). As can been seen in Fig. 1 A, addition of TM to a suspension culture of Nicotiana tabacum in the early log phase (3 d after subculture) caused an inhibition of growth. Growth as determined by the percentage of packed cells correlated fairly well with the increase in the number of cells up to the fifth day after subculture. As based on Trypan-blue exclusion the viability of the cells did not seem to be influenced by the TM concentrations used. This may also be obvious from the fact that in the presence of 0.2 ~g. m l - t TM, the culture started to proliferate again. The reversibility of the TM inhibition could also be demonstrated directly after resuspension of TMblocked cells in fresh medium. Proliferation resumed in about 2 d (data not shown). The inhibition of growth in the presence of TM was accompanied by a decrease in DNA content within 24 h. Protein synthesis on the other hand was not affected (Fig. 1 B, C). Direct measurement of the DNA and protein synthesis after labelling with
Metabolic labelling of cells. A 5-ml aliquot of cells was removed at various days of cultivation from the culture and pulsed under the same growing conditions with 1,9.105 Bq [CH3 - 3H]thymidine (Spec. activity 14.8 GBq.mmo1-1) or 9,5.104 Bq [35S]methionine (Spec. activity 0.37 TBq. mmol 1). Initial rates were calculated from time courses of incorporation of radioactivity into TCA-insoluble material. Measured aliquots were removed and stopped with 20% TCA. After centrifugation, the pellet was washed twice with 2 ml 10% TCA and once with 4 ml methanol. The pellet was resuspended in 0.1% sodium dodecyl sulfate (SDS) and the radioactivity measured. For incorporation of D-[1-14C]glucosamine (3,77" 104 Bqm l - 1 ; spec. activity 2.1 GBq. m m o l - 1) or D-[2-3H]mannose (3.77-104 Bq. m l - t ; spec. activity 0.5 TBq .mmol- 1), cells were used which had been subcultivated for at least 10 weeks in liquid medium with glucose as carbon source instead of sucrose. This was supposed to give better-defined conditions of the medium with respect to uptake and incorporation of the label, since uncontrolled sucrose breakdown by cell-wall-associated invertase is eliminated. For the experiment, cells were washed
30 -~20 IDI U)
~
"ID
A
/
200
Fig. 1 A-C. Effect of tunicamycin on
B 10
2~00 D o
t3
2
g
5
tt3
5
50
:I2 I---
m 3
.< Z Cl
O nr O
2~ Z.; 72' TIME (h)
96'
I-O n," 0_
,.; 7? & TiME [h)
C
"/ I
i
2Z. h~8 72 9; TIME (h)
growth (A), D N A (B), and protein (C) content in suspension cultures from Nicotiana tabacum. Tunicamycin (0.25 gg. m1-1 o o or 1 Ixg.m1-1 9 9 was added 3 d after subculture to a loggrowing culture; 9 . control. Measured aliquots were removed for determination of the various parameters as described in Materials and methods. The protein synthesized is the sum of the amount of protein within the cells plus that secreted into the medium. TM was added at time t = 0
C. Ettlinger et al. : Cell-cycle arrest by tunicamycin 2C
103 Fig, 2 A - C . Effect of tunicamycin on growth (A), D N A (B), and protein synthesis (C) in cultured cells from C a t h a r a n t h u s rosea. Tunicamycin was added at t = 0 3 d after subculture; 9 9 control, o o 0.1 gg-ml -~, 9 9 0.25 pg-m1-1. For D N A synthesis a 5-ml aliquot was taken at various times and pulsed with thymidine and a time course of incorporation of the radioactivity into D N A was followed. Numbers mean initial rates, expressed as cpm per 30 min
A
ol
% • u
9
o
~2
R lc
10F C
~o LU
o
[12 O
P l a n t a 9 Springer-Verlag 1986
Cell-cycle arrest of plant suspension cultures by tunicamycin C. Ettlinger, J. Schindler and L. Lehle Fakult/it ffir Biologie und Vorklinische Medizin, Universit/it Regensburg, Universitfitsstrasse 31, D-8400 Regensburg, Federal Republic of Germany
Abstract. The effect of tunicamycin, an inhibitor
of N-glycosylation of proteins, on growth and on synthesis of D N A and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1-1 gg. ml-1 tunicamycin, cell division and D N A synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear D N A content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the GI phase, as was recently postulated for animal and yeast cells. Key words: Catharanthus (cell cycle) - Cell cycle - Glycoprotein - Nicotiana (cell cycle) - Tunicamycin.
Introduction
Our understanding of the biochemical events of the eucaryotic cell and division cycle is far from complete. Although the pioneering experiments leading to the concept of four defined phases, G1, S, G2 and M, of the division cycle were made on a plant system (root tips of Vicia faba, Howard and Pelc 1951, 1953), cell cultures of higher plants have received less attention than animal cells (for reviews' see Gould 1984; Bryant 1976). There is evidence that the G1 and G2 phases serve as principal control points. In many merAbbreviations: TCA = trichloroacetic acid; TM = tunicamycin
istems, carbohydrate starvation leaves cells in GI or G2 (Van't H o f 1973). Deprivation of auxin from carrot cell culture leads to arrest in G1 (Nishi et al. 1977), and also cytokinin, gibberellin or abscisic acid seem to have control functions (for review, see Evans 1983; Nagl 1972). With root meristems' it was demonstrated that initiation of cell differentiation occurs in either GI or G2 (Roberts 1976). Further, the rate-limiting effect of temperature on growth in suspension cultures would also appear to be exerted during G1 (Gould 1977). Studies in animal cells (Pardee 1974; Campisi et al. 1982) and yeast (Hartwell 1974) have shown that the major regulatory events leading to proliferation in these organisms occur within a limited period during the G1 phase: cells beyond the socalled "restriction point" or " s t a r t " become committed to another cell cycle. More recently, investigations with cell lines from yeast (Arnold and Tanner 1982), Burkitt lymphoma (Nishikawa et al. 1980) and rat hepatoma (Savage and Baur 1983) have indicated that protein glycosylation is needed for transition from G1 to S. The present report extends this observation to higher-plant cells, and suggests that the phenomenon might have general relevance for all eucaryotic cells. It will be shown that tunicamycin, an inhibitor of N-glycosylation, arrests suspension cultures from Catharanthus at phase GI.
Materials and methods Material. Cells were grown in liquid suspension culture: Nicotiana tabacum in LS-medium (Linsmaier and Skoog 1965)
supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2 mg -1-1) and kinetin (0.03 mg-1 - 1); Catharanthus rosea in MSmedium (Murashige and Skoog 1962) in the presence of 2,4-D (0.44 mg ml 2). Cells were cultivated in the dark on a gyratory shaker at 110 rpm and 29 ~ C and subcultivated every week.
102
C. Ettlinger et al. : Cell-cycle arrest by tunicamycin
Methods of cell-cycle analysis. The D N A content was determined essentially according to Adams (1980) from 5-ml culture aliquots which were washed twice with 5 ml 0.85% NaC1 and once with 5% trichloroacetic acid (TCA). Calf thymus D N A was used as standard. Protein from cells was determined following the procedure of Lowry et al. (1951) after washing cells with 0.85% NaC1 and extracting it with 2 N N a O H at 85~ for 100 min. Aliquots of the clear supernatant were diluted with water and assayed. Protein content of the medium was determined after removal of cells by centrifugation from a measured aliquot. Nuclear D N A content of Feulgen-stained squashes was measured with a Zeiss scanning cytophotometer 05 (Zeiss, Oberkochem, F R G ) connected via CAMAC-crate to a Dietz 621 x 2 minicomputer (Dietz, FRG) as described by de Vries and Schindler (1984). Feulgen staining was carried out with p-rosanilin (Merck, Darmstadt, FRG) after strictly controlled fixation and acid hydrolysis. The cell culture (1 ml) was fixed twice for 20 min with 5 ml ethanol: acetic acid (3 : 1 ; v/v), applied to a slide and squashed. After hydrolysis in 1 N HC1 for 10 rain at 60 ~ C the specimens were stained for 3 h. Absorption of metaphase or telophase nuclei was equated with D N A content of G2-phase cells. To obtain the mitotic index (percentage number of mitotic figures divided by total nuclei counted) about 1500 cells were evaluated from the same preparation.
once in fresh medium without glucose and resuspended in the same medium. Tunicamycin (TM) was purchased from Calbiochem (La Jolla, Cal., USA) and was added as a filter-sterilized, aqueous solution after dissolving it in a small amount of 0.1 N N a O H and subsequent adjustment to pH 7.2. Controls contained the same aliquot of liquid without TM.
Results
Effects of tunicamycin on growth, and on synthesis o f D N A and protein. Tunicamycin has been shown to inhibit N-glycosylation of proteins by preventing the first reaction of the dolichol pathway, the transfer of N-acetylglucosamine 1-phosphate to the lipid intermediate carrier (Lehle and Tanner 1976; Takatsuki et al. 1975). As can been seen in Fig. 1 A, addition of TM to a suspension culture of Nicotiana tabacum in the early log phase (3 d after subculture) caused an inhibition of growth. Growth as determined by the percentage of packed cells correlated fairly well with the increase in the number of cells up to the fifth day after subculture. As based on Trypan-blue exclusion the viability of the cells did not seem to be influenced by the TM concentrations used. This may also be obvious from the fact that in the presence of 0.2 ~g. m l - t TM, the culture started to proliferate again. The reversibility of the TM inhibition could also be demonstrated directly after resuspension of TMblocked cells in fresh medium. Proliferation resumed in about 2 d (data not shown). The inhibition of growth in the presence of TM was accompanied by a decrease in DNA content within 24 h. Protein synthesis on the other hand was not affected (Fig. 1 B, C). Direct measurement of the DNA and protein synthesis after labelling with
Metabolic labelling of cells. A 5-ml aliquot of cells was removed at various days of cultivation from the culture and pulsed under the same growing conditions with 1,9.105 Bq [CH3 - 3H]thymidine (Spec. activity 14.8 GBq.mmo1-1) or 9,5.104 Bq [35S]methionine (Spec. activity 0.37 TBq. mmol 1). Initial rates were calculated from time courses of incorporation of radioactivity into TCA-insoluble material. Measured aliquots were removed and stopped with 20% TCA. After centrifugation, the pellet was washed twice with 2 ml 10% TCA and once with 4 ml methanol. The pellet was resuspended in 0.1% sodium dodecyl sulfate (SDS) and the radioactivity measured. For incorporation of D-[1-14C]glucosamine (3,77" 104 Bqm l - 1 ; spec. activity 2.1 GBq. m m o l - 1) or D-[2-3H]mannose (3.77-104 Bq. m l - t ; spec. activity 0.5 TBq .mmol- 1), cells were used which had been subcultivated for at least 10 weeks in liquid medium with glucose as carbon source instead of sucrose. This was supposed to give better-defined conditions of the medium with respect to uptake and incorporation of the label, since uncontrolled sucrose breakdown by cell-wall-associated invertase is eliminated. For the experiment, cells were washed
30 -~20 IDI U)
~
"ID
A
/
200
Fig. 1 A-C. Effect of tunicamycin on
B 10
2~00 D o
t3
2
g
5
tt3
5
50
:I2 I---
m 3
.< Z Cl
O nr O
2~ Z.; 72' TIME (h)
96'
I-O n," 0_
,.; 7? & TiME [h)
C
"/ I
i
2Z. h~8 72 9; TIME (h)
growth (A), D N A (B), and protein (C) content in suspension cultures from Nicotiana tabacum. Tunicamycin (0.25 gg. m1-1 o o or 1 Ixg.m1-1 9 9 was added 3 d after subculture to a loggrowing culture; 9 . control. Measured aliquots were removed for determination of the various parameters as described in Materials and methods. The protein synthesized is the sum of the amount of protein within the cells plus that secreted into the medium. TM was added at time t = 0
C. Ettlinger et al. : Cell-cycle arrest by tunicamycin 2C
103 Fig, 2 A - C . Effect of tunicamycin on growth (A), D N A (B), and protein synthesis (C) in cultured cells from C a t h a r a n t h u s rosea. Tunicamycin was added at t = 0 3 d after subculture; 9 9 control, o o 0.1 gg-ml -~, 9 9 0.25 pg-m1-1. For D N A synthesis a 5-ml aliquot was taken at various times and pulsed with thymidine and a time course of incorporation of the radioactivity into D N A was followed. Numbers mean initial rates, expressed as cpm per 30 min
A
ol
% • u
9
o
~2
R lc
10F C
~o LU
o
[12 O
