Printed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 00144827
Experimental Cell Research 104 (1977) 389-398
TEMPERATURE-SENSITIVE WITH
ALTERED
HAMSTER
MEMBRANE
CELL
LINE
PROPERTIES
H. CERI and J. A. WRIGHT Department of Microbiology, Winnipeg, Manitoba,
University of Manitoba, R3T 2N2
Canada,
SUMMARY The altered properties of a concanavalin A-resistant Chinese hamster ovary cell line with obvious temperature-sensitive growth properties is described. The variant cell line, P-7, was shown to have a higher efftciency of colony formation than the parental wild-type population after treatment with various concentrations of concanavaiin A (ConA). The variant cells had the properties of a temperature-sensitive cell line as judged by growth studies performed on solid surfaces or in suspension culture at the permissive (34°C) and non-permissive (39°C) temperatures; by colony efficiency determinations performed at 34°C and 39°C; and by the altered ability to incorporate DNA, RNA, and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for changes in the membrane properties of P-7 cells included: a reduced agghttinability in the presence of ConA, an altered cellular morphology on solid surfaces, an enhanced sensitivity to the toxic effects of membrane-active agents, altered and temperature-sensitive adhesiveness properties, and a reduced ability to bind labelled ConA.
Concanavalin A (ConA) is a lectin which interacts with polysaccharides and glycoproteins having (Y-D glucopyranosyl, a-D mannopyranosyl and related cell surface structures [l]. ConA is normally toxic for mammalian cells cultured in vitro [2,3], and has been used as a selective agent in cell culture for the isolation of Chinese hamster ovary (CHO) cell lines which are resistant to the cytotoxic effects of the lectin and show an enhanced sensitivity to a number of drugs [2]. In a brief report we have shown that ConA can be used as a tool in cell culture to obtain hamster cell lines which exhibit reduced plating efficiencies at 39°C relative to 34°C [4]. Since ConA interacts with cell surface structures involved in growth regulation [5] it seemed possible that lectin-resistant variants with altered surface membranes and temperature-
sensitive (ts) growth properties may be obtained in cell culture. Also, the cell surface is centrally involved in the regulation of growth [6] and variants containing both altered membranes and growth properties could eventually be useful in attempts to link growth regulatory mechanisms with changes in membrane structure and function. The purpose of this communication is to describe the altered growth properties of a ConA-resistant, ts variant and to provide some evidence for altered surface membranes. MATERIALS
AND METHODS
Cells and growth conditions Chinese hamster ovary (CHO) cells were grown as suspension and monolayer cultures at 34°C or 39°C in a-minimal essential medium (Flow Laboratories, Inc.) supplemented with antibiotics and 10% (vol/vol) Exp
Cell
Res 104 (1977)
390
Ceri and Wright Incorporation of radioactive precursors The radioactive compounds were purchased from AmershamDearle. Incorporation of [6-3H]TdR, [VH]uridine and [4,VH]L-leucine into acid-precipitable material was measured for a 15 min pulse in cell susuensions maintained in growth medium at 34°C or 39°C. The cells were collected by filtmtion onto glass fibre filters (Gelman no. 61630), washed with phosphate-buffered saline (PBS), incubated in ice-cold 10% trichloroacetic acid (TCA) for 10 mitt, washed with 70% ethanol and treated as previously reported [I 11. The various labelled precursors were added during the pulse to a final concentration of 1.0 &i/ml.
Cell agglutination assay
Id4
50
Fig. 1. Abscissa:
100
150
200
250
300
ConA @g/ml); ordinate: rel. colony-
forming ability. Effects of various concentrations of ConA treatment on the colony-forming ability of wild-type (0) and CR-7cells (A). Cells were incubated at 34°C.
Cells growing exponentially in suspension culture at 34°C were collected by centrifugation, washed with PBS, and suspended in 0.154 M NaCl solution at a concentration of 2~ lo6 cells/ml. For the agglutination assay 0.5 ml of the cell suspension and 0.5 ml of a 0.154 M NaCl solution containing varying concentrations of ConA were added to a 10x35 mm tissue culture dish. The ConA-treated suspension was slowly agitated at room temperature for 10 min and then examined with a light microscope for cell clumping. Agglutination was scored qualitatively on a scale of to ++++ (no agglutination to virtually complete agglutination).
Cell detachment studies fetal bovine serum (Reheis Chemical Co.) as previously reported [7]. Plating efficiencies were determined by standard techniques [8]. The cells gave negative tests for Mycoplasma contamination as judged by plating methods [9]. CR-7 is a cloned CHO cell line isolated from an uncloned population of ConAresistant cells obtained by a selection procedure detailed elsewhere [S]. In brief an uncloned ConAresistant population of CHO cells was obtained after 10 uassanes of a non-mutaaenized wild-tvne aonulation at 3;i”C in growth medium containing 46 pg/ml ConA; CR-7 is one of the subclones selected from the mixed. population of ConA-resistant cells and in preliminary exneriments the variant cells exhibited a reduced ability to form colonies in normal growth medium at 39°C comnared with 34°C. Analvsis of wildtype and CR-7 karydtypes was performed-on cell cultures previously treated with 0.6 pg/ml colchicine for 3 h. Karyological preparations were obtained by standard techniques [lo] and the chromosome complements were estimated by examining 60 phase contrast photomicrographs of each cell type. Both the wild-type and CR-7population exhibited a modal chromosome number of 21 with approx. 80% of the CR-7 cells and 95 % of the wild-&me cells exhibitine a chromosome complement of 20121 or 22. Approxy 12% of the V-7 cells and 3 % of the wild-type nonulation contained less than 20 chromosomes/cell and 8 % of CR-7 cells and 2% of the wild type had more than 22 chromosomes/cell. Exp Cell Res 104 (1977)
Wild-tvne and CR-7cells were mated at 6~ lo4 cells/ml on 15;;60 mm plastic tissue-culture plates and .incubated at 34°C or 39°C for 24 h in a CO, and humiditv controlled incubator. To carry out ceil detachment studies f121 the medium was removed: the cells were washedwiih PBS and incubated at 34°C or 39Y.Zwith 2 ml of PBS containina 0.03% trvnsin. At various time intervals the buff& was removed, cell aggregates were dispersed by pipetting, and the number of cells in suspension was counted with a cell counter (Coulter Electronics Co.).
Estimation of cell surface area Cells were removed from tissue culture dishes with PBS containing 0.15 % trypsin; the cell diameters were determined on at least 400 cells of each type with a light microscope fitted with a micrometer eyepiece. The average cell diameter was used to calculate [13] the cell surface area assuming the cells were smooth spheres.
ConA binding assay Cells were grown at 34°C on 15x60 mm plastic tissue culture mates to oartial contluencv. In orenaration for the binding experiment the plates were incubated at 0°C for 5 min [14]. The cells were then washed with ice-cold 0.154 M sodium chloride solution and in-
ts variant with altered membrane properties
with PBS containing 0.15% trypsin and passing the suspension through a cell counter (Coulter Electronics, Co.). Since the number of cells/plate and the amount of l&tin bound/plate could be determined the number of molecules of ConA bound/cell was calculated assuming a molecular weight of 110008 for the lectin [5]. A 5 min binding time was chosen because preliminary experiments indicated that maximum binding occurred within the 5 min period. Also the binding of labelled ConA was sugar specific since the amount of binding at all concentrations of ConA tested in the presence of 0.15 M a-methyl-u-mannoside was 15% or less of the binding which normally occurred in the absence of the hapten. The quantity of binding in the presence of the sugar was considered to be non-specific and was routinely monitored and subtracted from the binding estimates obtained in the absence of the inhibitor.
(I
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(q& 20
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RESULTS
Fig. 2. Abscissa: time (hours); ordinate: (a, b) ceils/ml; (c. d) cells/dish. Growth curves of the wild type at 34°C (0) and 39°C (0) and the F-7 cells at 34°C (A) and 39°C (A) in suspension culture (a, b) and on plastic tissue culture plates (c, d). In preparation for the experiments the cell lines were grown at 34°C; split into two separate cultures; and one member of each pair was shifted to 39°C while the other was kept at 34°C.
cubated for 5 min at 0°C in PBS containing the appropriate concentrations of [3H]ConA (New England Nuclear) in a final volume of 2.0 ml. The cells were then washed five times with ice-cold 0.154 M sodium chloride and solubilized for 60 min in 10% Triton X-100 (J. T. Baker Chemical Co.) at 37°C. Thedigested samples were added to an Aquasol cocktail (New England Nuclear) and counted in a liquid scintillation counter. The number of cells/tissue culture plate was determined by removing cells from untreated control plates
Resistance to ConA Wild-type and CR-7 cells were exposed to growth medium without serum but supplemented with various concentrations of ConA at 34°C for 48 h. The medium containing lectin was then replaced with fresh growth medium containing serum and without lectin. Ten days later the colonies that formed were stained with methylene blue [8]. It is apparent in fig. 1 that the CR-7 cells were less sensitive to the toxic effects of ConA when compared with the wild-type population. For example, the wild-type cells plated at an efficiency of about 1% after a treatment with 100 pg/ml ConA
Table 1. Plating efficiency a on 16 oz. culture bottles at various cell concentrations Cell line
No. of cells
No. of colonies (34°C)
Wild-type CR-7 CR-7 CR-7 CR-7 CR-7 CR-7
1Xlol 1x103 1x104 1x1@ 5x 103 1x106 5x 10s
762 701 ND ND ND ND ND
P.E. at 34°C 0.76 0.70 ND ND ND Ei
No. of colonies (39°C) 810 None None 2 38 105 2 260
P.E. at 39°C 0.81 2x 7.6x 1.1x 4.5x
10-S 1O-s 10-d 10-d
ND, not determined. (1 Surviving fraction of cells plated as determined by colony-forming ability. 26-761813
Exp Cell Res 104 (1977)
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Ceri and Wright
20
40
60
60
100
J---%
Fig. 3. Abscissa: time after temperature shift up; ordinate: (a) cpm/sample; (b) cell no./ml culture. A, TdR;
0, uridine; Cl, leucine. (a) Incorporation of labelled precursors into acidprecipitable material. Precursor incorporation into 5 ml samples of P-7 cells was measured in suspension culture durine a 15 min nuke before and after a temnerature shift from 34”C-to 39°C; (b) growth curveL at 39°C of the P-7 cells used in the incorporation study presented in (a).
whereas the plating efficiency of the variant population was not significantly affected by a treatment with the same concentration of lectin . Growth properties The P-7 and wild-type populations were tested for temperature sensitivity by determining growth rates at 34°C and 39°C on plastic tissue culture plates and in suspension culture. At the permissive temperature Exp Cell Res 104 (1977)
(34°C) both cell populations grew well (fig. 2); on plates CR-7 and wild-type cells exhibited doubling times of 18 to 20 h (fig. 2c, d) and in suspension culture the doubling times were 26 h and 22 h respectively (fig. 2a, 6). However, raising the temperature from 34°C to 39°C produced a dramatic difference in the growth ability exhibited by the two cell types (fig. 2). The wild-type cells continued to increase in cell number with a doubling time of 16 to 18 h on plates or in suspension culture (fig. 2b, d). It may be appropriate to mention that the wild-type cells used in these growth experiments had been maintained at the permissive temperature for a period of 6 months before the experiments were performed. In contrast to the control culture, the number of P-7 cells increased approx. 1.5 times on plates and less than 1.5 times in suspension culture within the first 24 h after the shift to the non-permissive temperature (fig. 2a, c). The F-7 cell number either remained approximately constant (fig. 2c) or actually declined (fig. 2a) while the variant cells were maintained at 39°C for a period of more than 100 h. Also a dye exclusion test for cell viability [15] was performed on the suspension cultures of wildtype and CR-7 cells maintained at 39°C for 48 h and it was found that more than 95% of the wild-type cells and less than 1% of the CR-7 population were actually viable. These results indicated that the CR-7 population had the properties of a ts variant. The ability to form colonies on a solid surface at 34°C and 39°C was determined by incubating cells at various cell concentrations in normal growth medium on 16 onz. Brockway bottles (which have about 90 cm* surface area for cell attachment) and calculating colony-forming efficiencies 10 days later by counting the number of colonies with 50 or more cells. It is apparent
ts variant with altered membrane properties
393
Fig. 4. Photographs of (a) F-7 cells and (b) wild-type CHO cells grown to about 80 % confluence on the sur-
face of a glass culture bottle at 34°C. Cells are magnified 160 times.
from table 1 that the CR-7 cells exhibited a reduced ability to form colonies at 39°C relative to 34°C. At the permissive temperature 76% of the wild-type cells and 70% of the CR-7 cells added to the bottles produced colonies. At the non-permissive temperature wild-type cells plated at an efficiency of 80%, whereas the variant cells showed a dramatic reduction in colonyforming ability. Although the CR-7 cells are markedly ts for colony-forming ability it is obvious (table 1) that there is a cell density effect since the fraction of CR-7 cells surviving at 39°C ranged from about 2x lo+’ when lo5 cells were plated to 4.5~ 10e4 when 5x 106cells were added to the culture bottle. The mechanism responsible for the cell density-dependent plating efficiency at 39°C is not understood and was not found at 34°C when various concentrations of CR-7 cells were added to plastic plates or glass bottles. However, the basis for this phenomenon may be similar to the mechanism of metabolic co-operation reported to occur among mammalian cells grown in culture [ 161.
Perhaps it should be noted that the. lectinresistant property and the ts growth properties described for the CR-7 cells are phenotypically stable since they have remained essentially unchanged during more than 100 generations of continuous culture in the absence of lectin at 34°C. Precursor incorporation at the nonpermissive temperature
To further characterize the ts effect observed with the CR-7 cells the ability to incorporate labelled TdR, uridine and leucine into acid-insoluble material during a 15 min pulse was studied as a function of time after raising the temperature of the suspension culture from 34°C to 39°C. Fig. 3 a indicates that the temperature change produced increases in the rates of TdR and leucine incorporation within the first few hours followed by a marked decline in the incorporation of these precursors over a period of about 4 days. Incorporation of uridine declined immediately after the temperature shift. Fig. 3 b shows that the cell number remained constant for the first 2 days after Exp Cell RPS 104 (1977)
394
Ceri and Wright
Table 2. Cell agglutination
in the presence
of ConA ConA cont. (/*g/ml) 0
50 250
500
750
loo0
A No trypsin treatment Wildtype ++ CR-7 ---
++++ ++
++++ +++
++++ ++++
B Trypsin treatment a Wildtype + CR-7 -
++ +
++++ ++
++++ ++++
a Cells were suspended in the presence of PBS containing 0.15 % trypsin for 15 min at room temperature, washed in 0.154 M NaCl 3 times and assayed as in Methods.
the increase in temperature and decreased during the last 2 days of the experiment. In control experiments with wild-type cells we observed increases in the rates of TdR, uridine and leucine incorporation during the first 2-4 h after temperature shift-up followed by a constant rate of precursor incorporation/cell during the 4 day period of the experiment. Also, the wild-type cell population exhibited the expected (fig. 2b) 16-18 h doubling time at 39°C. These results suggest that the ability to carry out macromolecular synthesis by the P-7 population at the non-permissive temperature is greatly reduced and resembles the reported ts properties of other mammalian cell variants maintained at the non-permissive temperature [ 171.
bottle or plate (fig. 4b); similar clumps of cells were not seen in cultures of wild-type cells (fig. 4a). We have also noticed that the variant cells usually appear to be oriented in a more disorganized or “criss-cross” pattern when compared with cultures of wild-type cells. These morphology differences may reflect actual changes in the composition or conformation of cell surface structures in the variant cells since it is known that modifications to the cell surface usually lead to changes in morphology [ 18, 19, 201. Cell agglutination of ConA
in the presence
ConA has the ability to agglutinate animal cells and this unusual property is often used as a tool or a probe to investigate cell surface differences [21]. The results of cell agglutination experiments in the presence of ConA are shown in table 2. Apparently higher ConA concentrations were required
Cell morphology
The appearance of wild-type and CR-7 cells at approx. 80% confluence is illustrated in fig. 4a, 6. One obvious difference between the two cell types is the frequent occurrence of large clumps or spheres of cells which form at the surface of the CR-7 cells attached to the solid support of a culture ExpCellRes 104 (1977)
2
4
6
6
0
02
06
IO
Fig. 5. Abscissa: (a) phenethyl alcohol (mM); (b) butyrate (mM); ordinate: ret. colony-forming ability. Effects of various concentrations of (u) phenethyl alcohol and (b) sodium butyrate on the colony-forming ability of 0, wild-type and A, P-7 cells grown at 34’C. Sodium butyrate was formed by adjusting the pH of a sample of butyric acid to 6.8 with NaOH.
ts variant with altered membrane properties
395
The CR-7 cells, which are of a more recent origin than the previously reported variants, were tested for the ability to form colonies 60 in the presence of the membrane active agents phenethyl alcohol [19, 221 and sodium butyrate [18, 231. In agreement with previous results on the “collateral sensitivity” of ConA-resistant cells [2, 241 the CR-7 population was found to be more sensitive to the toxic effects of these agents 40 20 30 IO (fig. 5). For example approx. 2 times (fig. Fig. 6. Abscissa: time (mitt); ordinate: % detached 5a) and 2.5 times (fig. 5b) the concentracells. Kinetics of detachment of the wild-type and CR-7 tions of phenethyl alcohol and sodium cells from a plastic tissue culture plate with PBS con- butyrate respectively were required to taining 0.03 % trypsin. 0, Wild-type cells and A, F-7 cells were incubated at 34°C for 24 h prior to the ex- reduce the relative plating efficiency of periment or, 0, wild-type cells and A, P-7 cells were wild-type cells to 1% when compared with incubated at 39°C for 24 h prior to the experiment. the concentrations needed to reduce the relative plating efficiency of the CR-7 cells to 1%. to agglutinate CR-7 cells than were required to agglutinate wild-type cells. For example, in the presence of 500 pug/ml lectin virtually 100% of the wild-type cells were agglutinated, whereas only 50% of the CR-7 cells were clumped. If ConA interacts with sugar residues attached to cell surface proteins during the agglutination assay perhaps removal of some of these glycoproteins with trypsin will reduce the ability of ConA to agglutinate the cells. As can be seen in table 2 higher concentrations of the lectin were required to agglutinate both cell types after trypsin treatment. Also, as expected, CR-7 cells still showed reduced agglutinability as compared with wild-type cells after trypsin treatment.
60.
Colony formation in the presence of membrane-active agents
ConA-resistant cells are often abnormally sensitive to agents which are known to interact with the cellular membrane [2].
Fig. 7. Abscissa: ConA @g/ml); ordinate: ConA molecules/~m*x lo+. Binding of [3H]ConA at 0°C per cell surface area at various concentrations of ConA. Three independent experiments were performed with 0, n , A, wild-type cells and three independent experiments were performed with 0, 0, A, P-7 cells. Exp Cell Res 104 (1977)
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Ceri and Wright
Cell detachment with trypsin Adhesion between the cell and its substratum can be quantitated by measuring the rate at which the cells are released from a solid surface with trypsin [12]. The enzyme releases cells by hydrolysing cell surface proteins involved in cell attachment. It is clear from fig. 6 that CR-7 cells are released from the surface at a greater rate than wild-type cells during treatment with 0.03% trypsin. For example within 15 min of trypsin treatment about 50% of the P-7 cells previously grown at 34°C for 24 h were released from the culture plate but only 5 % of the wild-type cells previously grown at either 34°C or 39°C for 24 h were released by trypsin during the first 15 min. Also note that the CR-7 detachment kinetics were markedly different when the cells were previously incubated for 24 h at 39°C as compared with 34°C; only 15% of the CR-7 cells incubated for 24 h at the non-permissive temperature prior to trypsin treatment were released within 15 min. This result suggests that the adhesive properties of CR-7 cells are different from wild-type cells and that this cell surface property is expressed as a ts function in the variant cells. ConA binding to intact cells Although transformed 3T3 cells agglutinate at lower concentrations of ConA than normal 3T3 cells various binding procedures have not shown differences in the amount of labelled lectin bound by the transformed and normal cell lines [25,26,27]. However, quantitative differences in the binding of labelled ConA was observed when the amount of endocytosis and non-specific binding was reduced by performing the assay procedure with [3H]ConA for 5 min at 0°C at pH 7.2 and 1.0 mM Mg2+ [14]. We have used the modified method to determine whether or not there is a significant Exp CeNRes 104 (1977)
difference in the ability of the wild type and the P-7 cells to bind ConA. In order to analyse the binding data in relation to cell surface area the average cell diameter was determined and used to calculate [ 131 the surface area of the wild-type (850 pm*) and the CR-7 cells (1 100 pm*). The amount of labelled ConA bound as a function of ConA concentration during a 5 min incubation at 0°C is shown in fig. 7. It is apparent that wild-type cells are capable of binding between 2.5 and 3 times more lectin as compared with P-7 cells. For example at 400 pug/ml lectin the wildtype population and the CR-7 cells bound about 12x 103 and 4x103 molecules of ConA/pm* surface area respectively. These results strongly suggest that the carbohydrate or the carbohydrate-containing components of the cell surface are altered in the ConA-resistant, ts variant. DISCUSSION Experimental evidence supports the view that the cell surface plays an important role in the regulation of cell growth. Attempts to link alterations in growth properties with changes in membrane structure and function should lead to a greater understanding of the membrane mechanisms involved in this fundamental biological process; mammalian variants expressing growth and cell surface alterations would be useful tools for these investigations. ConA often modifies the growth characteristics of cells in culture [5], probably interacts with the cell surface [21], and can be used to obtain lectinresistant cell lines [2]. Therefore it seemed possible that ConA-resistance could result from membrane changes that would not significantly affect the proliferation of cells at the selection temperature (34°C) but would alter the growth characteristics at
ts variant with altered membrane properties
some other, non-permissive, temperature (39°C). These lectin-resistant cells would exhibit ts growth properties and altered surface membranes. Clearly the lectin-resistant hamster cell line, CR-7fulfills the above criteria. The variant cells are obviously ts for growth as judged by proliferation studies and colony formation determinations carried out at 34°C and 39°C and by the reduced ability to incorporate DNA, RNA and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for alterations in the membrane properties of CR-7 cells include: reduced agglutinability in the presence of ConA, altered cellular morphology on solid surfaces, enhanced sensitivity to the toxic effects of membrane-active agents, altered ts adhesiveness properties, and a reduced ability to bind labelled ConA/cell surface area. We are now comparing the surface membranes of the variant and wild-type cells in an attempt to identify the modified structure(s) responsible for the variant phenotype. The reduced binding of labelled ConA suggests that the cell surface carbohydratecontaining structures of the variant cells may be altered. Some preliminary experiments with surface protein labelling techniques suggest that there may be membrane glycoprotein modifications in the CR-7cells. However, further studies are required to establish this point. There are still questions to be answered about the genetic basis for the complex phenotype of the CR-7cells. For example, is the variant phenotype the result of a true mutation, i.e. a stable change in the base sequence of the DNA as compared with a stable shift in gene expression? This is a diffkult question to answer and is a problem encountered with all mammalian somatic cell variants isolated in cell culture unless a
397
structural alteration in a gene product can be described [ll, 281. Examples of drugresistant somatic cell variants with altered gene products can be found in a recent review by Siminovitch [29]. Fortunately Chu [30] has suggested some criteria that would be useful in attempting to decide whether or not a variant phenotype is the result of a mutational event. In the present study one of the proposed guidelines is observed, i.e. retention of a stable phenotype in the absence of a selective agent. However, this point is consistent with both the mutational and epigenetic origin of somatic variation and this criterion alone does not constitute a proof for either mechanism. If we assume that the CR-7phenotype has resulted from a mutational event then we will eventually have to decide if the complex phenotype observed is the result of pleiotropic effects of a single mutation or are the consequence of multiple mutations. Support for the idea that a single event may be involved comes from our observations that there is a general correlation between the appearance of ConA resistance and the ts phenotype [4]; also more than 200 ConAresistant clones were recently tested for the ts property and at least 90 % of these freshly isolated variants exhibited varying degrees of temperature sensitivity. Furthermore recent preliminary experiments with three independently isolated ConA-resistant, ts variant lines have indicated that they also share the complex CR-7 phenotype outlined in this communication. Although the precise nature of the genetic events responsible for the complex CR-7 phenotype is not yet fully understood there are good indications that ConA-resistant, ts variants will be useful biological tools for studies aimed at gaining better understanding of the role played by the surface membrane in growth regulation. Exp Cell Res 104 (1977)
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Financial support for the study came from a grant to J. A. W. provided by the NCR of Canada. Aid provided by Dr I. Suzuki was also greatly appreciated. H. C. is the recipient of an NRC of Canada Scholarship.
REFERENCES 1. Goldstein, I, J & Staub, A M, Immunochemistry 7 (1970) 315. 2. kigdt, J A, J cell biol56 (1973) 666. 3. Storrie. B. J cell bio162 (1974) 247. 4. Wright; J A, Can j microbial 21 (1975) 1650. 5. Sharon, N & Lis, H, Science 177 (1972) 949. 6. Wallach, D F H, Proc natl acad sci US 61 (1968) 868. 7. Wright, J A & Lewis, W H, J celi physio183 (1974) 437. 8. Elkin, M M & Whitmore, G F, The radiobiology of cultured mammalian cells, p. 594. Gordon and Beach, New York (1%7). 9. Crawford, Y E, A laboratory guide to the mycoplasma of human origin, 2nd edn. Naval Medical Research Unit no. 4. Great Lakes. Ill. 60088, USA. 10. Rothfels, K H & Siminovitch, L, Stain technol 33 (1958) 73. 11. Lewis, W H & Wright, J A, Biochem biophys res commun 60 (1974) 926. 12. Rouyssegur, J M & Pastan, I, Proc natl acad sci US 73 (1976) 544. 13. Collard, J G & Temmink, J H M, J cell sci 19 (1975) 21. 14. Noonan, K D & Burger, M M, J biol them 248 (1973) 4286.
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15. Phillips, H J, Tissue culture methods and applications (ed P F Kruse, jr & M K Patterson, jr) p. 406. Academic Press, New York and London (1973). 16. Van Zeeland, A A, Van Diggelen, M C E & Simons, J W I M, Mutation res 14 (1972) 355. 17. Thompson, L H, Mankovitz, R, Baker, R M, Wright, J A, Till, J E, Siminovitch, L & Whitmore, G F, J cell physiol78 (1971) 43 1. 18. Wright, J A, Exp cell res 78 (1973) 456. 19. Wright, J A, Ceri, H & Lewis, W H, Nature new bio1244 (1973) 84. 20. Henneberry, R C, Fishman, P H & Freese, E, Cell 5 (1975) 1. 21. Oseroff, A R, Robbins, P W & Burger, M M, Ann rev biochem 42 (1973) 647. 22. Brossmer, R, Bohn, B & Schlicker, H, FEBS lett 35 (1973) 191. 23. Simmons, J L, Fishman, P H, Freese, E & Brady, R 0, J cell bio166 (1975) 414. 24. Till, J E, Baker, R M, Brunette, D M, Ling, V, Thompson, L H & Wright, J A, Fed proc 32 (1973) 29. 25. Cline. M J & Livinaston. D C. Nature new biol232 (1971) 155. 26. Ozanne, B & Sambrook, J, Nature new biol 232 (1971) 156. 27. Ardnt-Jovin. D J &Beta. P. J virol8 (1971) 716. 28. Wright, J A, Biochem-biophys res ‘commun 66 (1975) 584. 29. Siminovitch, L, Cell 7 (1976) 1. 30. Chu, E H Y, Genetics 78 (1974) 115. Received June 17, 1976 Accepted August 18, 1976
Experimental Cell Research 104 (1977) 389-398
TEMPERATURE-SENSITIVE WITH
ALTERED
HAMSTER
MEMBRANE
CELL
LINE
PROPERTIES
H. CERI and J. A. WRIGHT Department of Microbiology, Winnipeg, Manitoba,
University of Manitoba, R3T 2N2
Canada,
SUMMARY The altered properties of a concanavalin A-resistant Chinese hamster ovary cell line with obvious temperature-sensitive growth properties is described. The variant cell line, P-7, was shown to have a higher efftciency of colony formation than the parental wild-type population after treatment with various concentrations of concanavaiin A (ConA). The variant cells had the properties of a temperature-sensitive cell line as judged by growth studies performed on solid surfaces or in suspension culture at the permissive (34°C) and non-permissive (39°C) temperatures; by colony efficiency determinations performed at 34°C and 39°C; and by the altered ability to incorporate DNA, RNA, and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for changes in the membrane properties of P-7 cells included: a reduced agghttinability in the presence of ConA, an altered cellular morphology on solid surfaces, an enhanced sensitivity to the toxic effects of membrane-active agents, altered and temperature-sensitive adhesiveness properties, and a reduced ability to bind labelled ConA.
Concanavalin A (ConA) is a lectin which interacts with polysaccharides and glycoproteins having (Y-D glucopyranosyl, a-D mannopyranosyl and related cell surface structures [l]. ConA is normally toxic for mammalian cells cultured in vitro [2,3], and has been used as a selective agent in cell culture for the isolation of Chinese hamster ovary (CHO) cell lines which are resistant to the cytotoxic effects of the lectin and show an enhanced sensitivity to a number of drugs [2]. In a brief report we have shown that ConA can be used as a tool in cell culture to obtain hamster cell lines which exhibit reduced plating efficiencies at 39°C relative to 34°C [4]. Since ConA interacts with cell surface structures involved in growth regulation [5] it seemed possible that lectin-resistant variants with altered surface membranes and temperature-
sensitive (ts) growth properties may be obtained in cell culture. Also, the cell surface is centrally involved in the regulation of growth [6] and variants containing both altered membranes and growth properties could eventually be useful in attempts to link growth regulatory mechanisms with changes in membrane structure and function. The purpose of this communication is to describe the altered growth properties of a ConA-resistant, ts variant and to provide some evidence for altered surface membranes. MATERIALS
AND METHODS
Cells and growth conditions Chinese hamster ovary (CHO) cells were grown as suspension and monolayer cultures at 34°C or 39°C in a-minimal essential medium (Flow Laboratories, Inc.) supplemented with antibiotics and 10% (vol/vol) Exp
Cell
Res 104 (1977)
390
Ceri and Wright Incorporation of radioactive precursors The radioactive compounds were purchased from AmershamDearle. Incorporation of [6-3H]TdR, [VH]uridine and [4,VH]L-leucine into acid-precipitable material was measured for a 15 min pulse in cell susuensions maintained in growth medium at 34°C or 39°C. The cells were collected by filtmtion onto glass fibre filters (Gelman no. 61630), washed with phosphate-buffered saline (PBS), incubated in ice-cold 10% trichloroacetic acid (TCA) for 10 mitt, washed with 70% ethanol and treated as previously reported [I 11. The various labelled precursors were added during the pulse to a final concentration of 1.0 &i/ml.
Cell agglutination assay
Id4
50
Fig. 1. Abscissa:
100
150
200
250
300
ConA @g/ml); ordinate: rel. colony-
forming ability. Effects of various concentrations of ConA treatment on the colony-forming ability of wild-type (0) and CR-7cells (A). Cells were incubated at 34°C.
Cells growing exponentially in suspension culture at 34°C were collected by centrifugation, washed with PBS, and suspended in 0.154 M NaCl solution at a concentration of 2~ lo6 cells/ml. For the agglutination assay 0.5 ml of the cell suspension and 0.5 ml of a 0.154 M NaCl solution containing varying concentrations of ConA were added to a 10x35 mm tissue culture dish. The ConA-treated suspension was slowly agitated at room temperature for 10 min and then examined with a light microscope for cell clumping. Agglutination was scored qualitatively on a scale of to ++++ (no agglutination to virtually complete agglutination).
Cell detachment studies fetal bovine serum (Reheis Chemical Co.) as previously reported [7]. Plating efficiencies were determined by standard techniques [8]. The cells gave negative tests for Mycoplasma contamination as judged by plating methods [9]. CR-7 is a cloned CHO cell line isolated from an uncloned population of ConAresistant cells obtained by a selection procedure detailed elsewhere [S]. In brief an uncloned ConAresistant population of CHO cells was obtained after 10 uassanes of a non-mutaaenized wild-tvne aonulation at 3;i”C in growth medium containing 46 pg/ml ConA; CR-7 is one of the subclones selected from the mixed. population of ConA-resistant cells and in preliminary exneriments the variant cells exhibited a reduced ability to form colonies in normal growth medium at 39°C comnared with 34°C. Analvsis of wildtype and CR-7 karydtypes was performed-on cell cultures previously treated with 0.6 pg/ml colchicine for 3 h. Karyological preparations were obtained by standard techniques [lo] and the chromosome complements were estimated by examining 60 phase contrast photomicrographs of each cell type. Both the wild-type and CR-7population exhibited a modal chromosome number of 21 with approx. 80% of the CR-7 cells and 95 % of the wild-&me cells exhibitine a chromosome complement of 20121 or 22. Approxy 12% of the V-7 cells and 3 % of the wild-type nonulation contained less than 20 chromosomes/cell and 8 % of CR-7 cells and 2% of the wild type had more than 22 chromosomes/cell. Exp Cell Res 104 (1977)
Wild-tvne and CR-7cells were mated at 6~ lo4 cells/ml on 15;;60 mm plastic tissue-culture plates and .incubated at 34°C or 39°C for 24 h in a CO, and humiditv controlled incubator. To carry out ceil detachment studies f121 the medium was removed: the cells were washedwiih PBS and incubated at 34°C or 39Y.Zwith 2 ml of PBS containina 0.03% trvnsin. At various time intervals the buff& was removed, cell aggregates were dispersed by pipetting, and the number of cells in suspension was counted with a cell counter (Coulter Electronics Co.).
Estimation of cell surface area Cells were removed from tissue culture dishes with PBS containing 0.15 % trypsin; the cell diameters were determined on at least 400 cells of each type with a light microscope fitted with a micrometer eyepiece. The average cell diameter was used to calculate [13] the cell surface area assuming the cells were smooth spheres.
ConA binding assay Cells were grown at 34°C on 15x60 mm plastic tissue culture mates to oartial contluencv. In orenaration for the binding experiment the plates were incubated at 0°C for 5 min [14]. The cells were then washed with ice-cold 0.154 M sodium chloride solution and in-
ts variant with altered membrane properties
with PBS containing 0.15% trypsin and passing the suspension through a cell counter (Coulter Electronics, Co.). Since the number of cells/plate and the amount of l&tin bound/plate could be determined the number of molecules of ConA bound/cell was calculated assuming a molecular weight of 110008 for the lectin [5]. A 5 min binding time was chosen because preliminary experiments indicated that maximum binding occurred within the 5 min period. Also the binding of labelled ConA was sugar specific since the amount of binding at all concentrations of ConA tested in the presence of 0.15 M a-methyl-u-mannoside was 15% or less of the binding which normally occurred in the absence of the hapten. The quantity of binding in the presence of the sugar was considered to be non-specific and was routinely monitored and subtracted from the binding estimates obtained in the absence of the inhibitor.
(I
.::i
20
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100 120
20
40
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20
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P
(q& 20
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RESULTS
Fig. 2. Abscissa: time (hours); ordinate: (a, b) ceils/ml; (c. d) cells/dish. Growth curves of the wild type at 34°C (0) and 39°C (0) and the F-7 cells at 34°C (A) and 39°C (A) in suspension culture (a, b) and on plastic tissue culture plates (c, d). In preparation for the experiments the cell lines were grown at 34°C; split into two separate cultures; and one member of each pair was shifted to 39°C while the other was kept at 34°C.
cubated for 5 min at 0°C in PBS containing the appropriate concentrations of [3H]ConA (New England Nuclear) in a final volume of 2.0 ml. The cells were then washed five times with ice-cold 0.154 M sodium chloride and solubilized for 60 min in 10% Triton X-100 (J. T. Baker Chemical Co.) at 37°C. Thedigested samples were added to an Aquasol cocktail (New England Nuclear) and counted in a liquid scintillation counter. The number of cells/tissue culture plate was determined by removing cells from untreated control plates
Resistance to ConA Wild-type and CR-7 cells were exposed to growth medium without serum but supplemented with various concentrations of ConA at 34°C for 48 h. The medium containing lectin was then replaced with fresh growth medium containing serum and without lectin. Ten days later the colonies that formed were stained with methylene blue [8]. It is apparent in fig. 1 that the CR-7 cells were less sensitive to the toxic effects of ConA when compared with the wild-type population. For example, the wild-type cells plated at an efficiency of about 1% after a treatment with 100 pg/ml ConA
Table 1. Plating efficiency a on 16 oz. culture bottles at various cell concentrations Cell line
No. of cells
No. of colonies (34°C)
Wild-type CR-7 CR-7 CR-7 CR-7 CR-7 CR-7
1Xlol 1x103 1x104 1x1@ 5x 103 1x106 5x 10s
762 701 ND ND ND ND ND
P.E. at 34°C 0.76 0.70 ND ND ND Ei
No. of colonies (39°C) 810 None None 2 38 105 2 260
P.E. at 39°C 0.81 2x 7.6x 1.1x 4.5x
10-S 1O-s 10-d 10-d
ND, not determined. (1 Surviving fraction of cells plated as determined by colony-forming ability. 26-761813
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20
40
60
60
100
J---%
Fig. 3. Abscissa: time after temperature shift up; ordinate: (a) cpm/sample; (b) cell no./ml culture. A, TdR;
0, uridine; Cl, leucine. (a) Incorporation of labelled precursors into acidprecipitable material. Precursor incorporation into 5 ml samples of P-7 cells was measured in suspension culture durine a 15 min nuke before and after a temnerature shift from 34”C-to 39°C; (b) growth curveL at 39°C of the P-7 cells used in the incorporation study presented in (a).
whereas the plating efficiency of the variant population was not significantly affected by a treatment with the same concentration of lectin . Growth properties The P-7 and wild-type populations were tested for temperature sensitivity by determining growth rates at 34°C and 39°C on plastic tissue culture plates and in suspension culture. At the permissive temperature Exp Cell Res 104 (1977)
(34°C) both cell populations grew well (fig. 2); on plates CR-7 and wild-type cells exhibited doubling times of 18 to 20 h (fig. 2c, d) and in suspension culture the doubling times were 26 h and 22 h respectively (fig. 2a, 6). However, raising the temperature from 34°C to 39°C produced a dramatic difference in the growth ability exhibited by the two cell types (fig. 2). The wild-type cells continued to increase in cell number with a doubling time of 16 to 18 h on plates or in suspension culture (fig. 2b, d). It may be appropriate to mention that the wild-type cells used in these growth experiments had been maintained at the permissive temperature for a period of 6 months before the experiments were performed. In contrast to the control culture, the number of P-7 cells increased approx. 1.5 times on plates and less than 1.5 times in suspension culture within the first 24 h after the shift to the non-permissive temperature (fig. 2a, c). The F-7 cell number either remained approximately constant (fig. 2c) or actually declined (fig. 2a) while the variant cells were maintained at 39°C for a period of more than 100 h. Also a dye exclusion test for cell viability [15] was performed on the suspension cultures of wildtype and CR-7 cells maintained at 39°C for 48 h and it was found that more than 95% of the wild-type cells and less than 1% of the CR-7 population were actually viable. These results indicated that the CR-7 population had the properties of a ts variant. The ability to form colonies on a solid surface at 34°C and 39°C was determined by incubating cells at various cell concentrations in normal growth medium on 16 onz. Brockway bottles (which have about 90 cm* surface area for cell attachment) and calculating colony-forming efficiencies 10 days later by counting the number of colonies with 50 or more cells. It is apparent
ts variant with altered membrane properties
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Fig. 4. Photographs of (a) F-7 cells and (b) wild-type CHO cells grown to about 80 % confluence on the sur-
face of a glass culture bottle at 34°C. Cells are magnified 160 times.
from table 1 that the CR-7 cells exhibited a reduced ability to form colonies at 39°C relative to 34°C. At the permissive temperature 76% of the wild-type cells and 70% of the CR-7 cells added to the bottles produced colonies. At the non-permissive temperature wild-type cells plated at an efficiency of 80%, whereas the variant cells showed a dramatic reduction in colonyforming ability. Although the CR-7 cells are markedly ts for colony-forming ability it is obvious (table 1) that there is a cell density effect since the fraction of CR-7 cells surviving at 39°C ranged from about 2x lo+’ when lo5 cells were plated to 4.5~ 10e4 when 5x 106cells were added to the culture bottle. The mechanism responsible for the cell density-dependent plating efficiency at 39°C is not understood and was not found at 34°C when various concentrations of CR-7 cells were added to plastic plates or glass bottles. However, the basis for this phenomenon may be similar to the mechanism of metabolic co-operation reported to occur among mammalian cells grown in culture [ 161.
Perhaps it should be noted that the. lectinresistant property and the ts growth properties described for the CR-7 cells are phenotypically stable since they have remained essentially unchanged during more than 100 generations of continuous culture in the absence of lectin at 34°C. Precursor incorporation at the nonpermissive temperature
To further characterize the ts effect observed with the CR-7 cells the ability to incorporate labelled TdR, uridine and leucine into acid-insoluble material during a 15 min pulse was studied as a function of time after raising the temperature of the suspension culture from 34°C to 39°C. Fig. 3 a indicates that the temperature change produced increases in the rates of TdR and leucine incorporation within the first few hours followed by a marked decline in the incorporation of these precursors over a period of about 4 days. Incorporation of uridine declined immediately after the temperature shift. Fig. 3 b shows that the cell number remained constant for the first 2 days after Exp Cell RPS 104 (1977)
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Table 2. Cell agglutination
in the presence
of ConA ConA cont. (/*g/ml) 0
50 250
500
750
loo0
A No trypsin treatment Wildtype ++ CR-7 ---
++++ ++
++++ +++
++++ ++++
B Trypsin treatment a Wildtype + CR-7 -
++ +
++++ ++
++++ ++++
a Cells were suspended in the presence of PBS containing 0.15 % trypsin for 15 min at room temperature, washed in 0.154 M NaCl 3 times and assayed as in Methods.
the increase in temperature and decreased during the last 2 days of the experiment. In control experiments with wild-type cells we observed increases in the rates of TdR, uridine and leucine incorporation during the first 2-4 h after temperature shift-up followed by a constant rate of precursor incorporation/cell during the 4 day period of the experiment. Also, the wild-type cell population exhibited the expected (fig. 2b) 16-18 h doubling time at 39°C. These results suggest that the ability to carry out macromolecular synthesis by the P-7 population at the non-permissive temperature is greatly reduced and resembles the reported ts properties of other mammalian cell variants maintained at the non-permissive temperature [ 171.
bottle or plate (fig. 4b); similar clumps of cells were not seen in cultures of wild-type cells (fig. 4a). We have also noticed that the variant cells usually appear to be oriented in a more disorganized or “criss-cross” pattern when compared with cultures of wild-type cells. These morphology differences may reflect actual changes in the composition or conformation of cell surface structures in the variant cells since it is known that modifications to the cell surface usually lead to changes in morphology [ 18, 19, 201. Cell agglutination of ConA
in the presence
ConA has the ability to agglutinate animal cells and this unusual property is often used as a tool or a probe to investigate cell surface differences [21]. The results of cell agglutination experiments in the presence of ConA are shown in table 2. Apparently higher ConA concentrations were required
Cell morphology
The appearance of wild-type and CR-7 cells at approx. 80% confluence is illustrated in fig. 4a, 6. One obvious difference between the two cell types is the frequent occurrence of large clumps or spheres of cells which form at the surface of the CR-7 cells attached to the solid support of a culture ExpCellRes 104 (1977)
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4
6
6
0
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Fig. 5. Abscissa: (a) phenethyl alcohol (mM); (b) butyrate (mM); ordinate: ret. colony-forming ability. Effects of various concentrations of (u) phenethyl alcohol and (b) sodium butyrate on the colony-forming ability of 0, wild-type and A, P-7 cells grown at 34’C. Sodium butyrate was formed by adjusting the pH of a sample of butyric acid to 6.8 with NaOH.
ts variant with altered membrane properties
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The CR-7 cells, which are of a more recent origin than the previously reported variants, were tested for the ability to form colonies 60 in the presence of the membrane active agents phenethyl alcohol [19, 221 and sodium butyrate [18, 231. In agreement with previous results on the “collateral sensitivity” of ConA-resistant cells [2, 241 the CR-7 population was found to be more sensitive to the toxic effects of these agents 40 20 30 IO (fig. 5). For example approx. 2 times (fig. Fig. 6. Abscissa: time (mitt); ordinate: % detached 5a) and 2.5 times (fig. 5b) the concentracells. Kinetics of detachment of the wild-type and CR-7 tions of phenethyl alcohol and sodium cells from a plastic tissue culture plate with PBS con- butyrate respectively were required to taining 0.03 % trypsin. 0, Wild-type cells and A, F-7 cells were incubated at 34°C for 24 h prior to the ex- reduce the relative plating efficiency of periment or, 0, wild-type cells and A, P-7 cells were wild-type cells to 1% when compared with incubated at 39°C for 24 h prior to the experiment. the concentrations needed to reduce the relative plating efficiency of the CR-7 cells to 1%. to agglutinate CR-7 cells than were required to agglutinate wild-type cells. For example, in the presence of 500 pug/ml lectin virtually 100% of the wild-type cells were agglutinated, whereas only 50% of the CR-7 cells were clumped. If ConA interacts with sugar residues attached to cell surface proteins during the agglutination assay perhaps removal of some of these glycoproteins with trypsin will reduce the ability of ConA to agglutinate the cells. As can be seen in table 2 higher concentrations of the lectin were required to agglutinate both cell types after trypsin treatment. Also, as expected, CR-7 cells still showed reduced agglutinability as compared with wild-type cells after trypsin treatment.
60.
Colony formation in the presence of membrane-active agents
ConA-resistant cells are often abnormally sensitive to agents which are known to interact with the cellular membrane [2].
Fig. 7. Abscissa: ConA @g/ml); ordinate: ConA molecules/~m*x lo+. Binding of [3H]ConA at 0°C per cell surface area at various concentrations of ConA. Three independent experiments were performed with 0, n , A, wild-type cells and three independent experiments were performed with 0, 0, A, P-7 cells. Exp Cell Res 104 (1977)
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Cell detachment with trypsin Adhesion between the cell and its substratum can be quantitated by measuring the rate at which the cells are released from a solid surface with trypsin [12]. The enzyme releases cells by hydrolysing cell surface proteins involved in cell attachment. It is clear from fig. 6 that CR-7 cells are released from the surface at a greater rate than wild-type cells during treatment with 0.03% trypsin. For example within 15 min of trypsin treatment about 50% of the P-7 cells previously grown at 34°C for 24 h were released from the culture plate but only 5 % of the wild-type cells previously grown at either 34°C or 39°C for 24 h were released by trypsin during the first 15 min. Also note that the CR-7 detachment kinetics were markedly different when the cells were previously incubated for 24 h at 39°C as compared with 34°C; only 15% of the CR-7 cells incubated for 24 h at the non-permissive temperature prior to trypsin treatment were released within 15 min. This result suggests that the adhesive properties of CR-7 cells are different from wild-type cells and that this cell surface property is expressed as a ts function in the variant cells. ConA binding to intact cells Although transformed 3T3 cells agglutinate at lower concentrations of ConA than normal 3T3 cells various binding procedures have not shown differences in the amount of labelled lectin bound by the transformed and normal cell lines [25,26,27]. However, quantitative differences in the binding of labelled ConA was observed when the amount of endocytosis and non-specific binding was reduced by performing the assay procedure with [3H]ConA for 5 min at 0°C at pH 7.2 and 1.0 mM Mg2+ [14]. We have used the modified method to determine whether or not there is a significant Exp CeNRes 104 (1977)
difference in the ability of the wild type and the P-7 cells to bind ConA. In order to analyse the binding data in relation to cell surface area the average cell diameter was determined and used to calculate [ 131 the surface area of the wild-type (850 pm*) and the CR-7 cells (1 100 pm*). The amount of labelled ConA bound as a function of ConA concentration during a 5 min incubation at 0°C is shown in fig. 7. It is apparent that wild-type cells are capable of binding between 2.5 and 3 times more lectin as compared with P-7 cells. For example at 400 pug/ml lectin the wildtype population and the CR-7 cells bound about 12x 103 and 4x103 molecules of ConA/pm* surface area respectively. These results strongly suggest that the carbohydrate or the carbohydrate-containing components of the cell surface are altered in the ConA-resistant, ts variant. DISCUSSION Experimental evidence supports the view that the cell surface plays an important role in the regulation of cell growth. Attempts to link alterations in growth properties with changes in membrane structure and function should lead to a greater understanding of the membrane mechanisms involved in this fundamental biological process; mammalian variants expressing growth and cell surface alterations would be useful tools for these investigations. ConA often modifies the growth characteristics of cells in culture [5], probably interacts with the cell surface [21], and can be used to obtain lectinresistant cell lines [2]. Therefore it seemed possible that ConA-resistance could result from membrane changes that would not significantly affect the proliferation of cells at the selection temperature (34°C) but would alter the growth characteristics at
ts variant with altered membrane properties
some other, non-permissive, temperature (39°C). These lectin-resistant cells would exhibit ts growth properties and altered surface membranes. Clearly the lectin-resistant hamster cell line, CR-7fulfills the above criteria. The variant cells are obviously ts for growth as judged by proliferation studies and colony formation determinations carried out at 34°C and 39°C and by the reduced ability to incorporate DNA, RNA and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for alterations in the membrane properties of CR-7 cells include: reduced agglutinability in the presence of ConA, altered cellular morphology on solid surfaces, enhanced sensitivity to the toxic effects of membrane-active agents, altered ts adhesiveness properties, and a reduced ability to bind labelled ConA/cell surface area. We are now comparing the surface membranes of the variant and wild-type cells in an attempt to identify the modified structure(s) responsible for the variant phenotype. The reduced binding of labelled ConA suggests that the cell surface carbohydratecontaining structures of the variant cells may be altered. Some preliminary experiments with surface protein labelling techniques suggest that there may be membrane glycoprotein modifications in the CR-7cells. However, further studies are required to establish this point. There are still questions to be answered about the genetic basis for the complex phenotype of the CR-7cells. For example, is the variant phenotype the result of a true mutation, i.e. a stable change in the base sequence of the DNA as compared with a stable shift in gene expression? This is a diffkult question to answer and is a problem encountered with all mammalian somatic cell variants isolated in cell culture unless a
397
structural alteration in a gene product can be described [ll, 281. Examples of drugresistant somatic cell variants with altered gene products can be found in a recent review by Siminovitch [29]. Fortunately Chu [30] has suggested some criteria that would be useful in attempting to decide whether or not a variant phenotype is the result of a mutational event. In the present study one of the proposed guidelines is observed, i.e. retention of a stable phenotype in the absence of a selective agent. However, this point is consistent with both the mutational and epigenetic origin of somatic variation and this criterion alone does not constitute a proof for either mechanism. If we assume that the CR-7phenotype has resulted from a mutational event then we will eventually have to decide if the complex phenotype observed is the result of pleiotropic effects of a single mutation or are the consequence of multiple mutations. Support for the idea that a single event may be involved comes from our observations that there is a general correlation between the appearance of ConA resistance and the ts phenotype [4]; also more than 200 ConAresistant clones were recently tested for the ts property and at least 90 % of these freshly isolated variants exhibited varying degrees of temperature sensitivity. Furthermore recent preliminary experiments with three independently isolated ConA-resistant, ts variant lines have indicated that they also share the complex CR-7 phenotype outlined in this communication. Although the precise nature of the genetic events responsible for the complex CR-7 phenotype is not yet fully understood there are good indications that ConA-resistant, ts variants will be useful biological tools for studies aimed at gaining better understanding of the role played by the surface membrane in growth regulation. Exp Cell Res 104 (1977)
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Financial support for the study came from a grant to J. A. W. provided by the NCR of Canada. Aid provided by Dr I. Suzuki was also greatly appreciated. H. C. is the recipient of an NRC of Canada Scholarship.
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15. Phillips, H J, Tissue culture methods and applications (ed P F Kruse, jr & M K Patterson, jr) p. 406. Academic Press, New York and London (1973). 16. Van Zeeland, A A, Van Diggelen, M C E & Simons, J W I M, Mutation res 14 (1972) 355. 17. Thompson, L H, Mankovitz, R, Baker, R M, Wright, J A, Till, J E, Siminovitch, L & Whitmore, G F, J cell physiol78 (1971) 43 1. 18. Wright, J A, Exp cell res 78 (1973) 456. 19. Wright, J A, Ceri, H & Lewis, W H, Nature new bio1244 (1973) 84. 20. Henneberry, R C, Fishman, P H & Freese, E, Cell 5 (1975) 1. 21. Oseroff, A R, Robbins, P W & Burger, M M, Ann rev biochem 42 (1973) 647. 22. Brossmer, R, Bohn, B & Schlicker, H, FEBS lett 35 (1973) 191. 23. Simmons, J L, Fishman, P H, Freese, E & Brady, R 0, J cell bio166 (1975) 414. 24. Till, J E, Baker, R M, Brunette, D M, Ling, V, Thompson, L H & Wright, J A, Fed proc 32 (1973) 29. 25. Cline. M J & Livinaston. D C. Nature new biol232 (1971) 155. 26. Ozanne, B & Sambrook, J, Nature new biol 232 (1971) 156. 27. Ardnt-Jovin. D J &Beta. P. J virol8 (1971) 716. 28. Wright, J A, Biochem-biophys res ‘commun 66 (1975) 584. 29. Siminovitch, L, Cell 7 (1976) 1. 30. Chu, E H Y, Genetics 78 (1974) 115. Received June 17, 1976 Accepted August 18, 1976
