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Experimental Cell Research 114 (1978) 217-227 A CORRELATION AND

BETWEEN

SPECIFIC

SURFACE

CONCANAVALIN

ALTERATIONS

A RESISTANCE

IN GROWTH

MEMBRANE-ASSOCIATED

AND

PROPERTIES

HOWARD CERI and JIM A. WRIGHT Department

of Microbiology,

University

of Manitoba,

Winnipeg, Manitoba,

Canada, R3T 2N2

SUMMARY Mammalian cells selected for resistance to concanavalin A (ConA) cytotoxicity exhibit modifications in some fundamental cellular properties. Three independently isolated ConA-resistant hamster cell lines exhibit a complex phenotype which includes: obvious temperature-sensitive growth properties; altered cellular morphology on solid surfaces; enhanced sensitivity to membrane-active agents such as phenethyl alcohol and sodium butyrate; altered lectin agglutination properties; modified adhesiveness to substratum properties; and defective lectin-receptor mobility characteristics. Selection of a revertant cell line which showed a near wild-type sensitivity to the cytotoxic effects of ConA also showed growth and membrane-associated properties that were very similar to parental wild-type cells. Somatic cell hybrids formed through the fusion of wildtype and lectin-resistant cells exhibited the ConA-sensitive phenotype, and possessed growth and membrane-associated properties that were very similar to pseudodiploid wild-type cells and control cultures of pseudotetraploid hybrid cells. The results presented in this communication support the view that ConA is an excellent selective agent for obtaining mammalian cells with altered growth and surface membrane properties and provides convincing evidence that the altered cellular properties exhibited by the lectin-resistant cell lines are directly related to ConA resistance.

The surface membrane is intimately in- characteristics and alterations in a number volved in the regulation of many important of membrane-associated properties [6]. cellular events. For example the cell sur- Therefore, it appeared that ConA resistance face plays an important role in the regula- was accompanied by modifications in some tion of intercellular communication, cell fundamental biological properties. Since growth, the immune response, differentia- most of the initial studies were carried out tion, and in viral oncogenesis [l-4]. Mam- with the CR-7 cell line it was important malian variants with altered surface mem- to investigate the relationship between branes provide a novel opportunity to in- ConA resistance and the complex phenovestigate the mechanisms involved in this type with other independently selected regulation. With this view in mind we have resistant and sensitive cell lines. In this isolated Chinese hamster ovary cells which paper we describe the cellular characterexhibit resistance to the cytotoxic effects of istics of a number of ConA-resistant, the plant lectin, ConA [5]. One of these cell sensitive and revertant cell lines and show lines (CR-7) has been investigated in detail that a correlation does occur between and exhibited a complex phenotype which the complex phenotype and ConA resistincluded temperature-sensitive growth ance. Exp Cell Rrs I14 (I 978)

Ceri and Wright

218

WILD TYPE POPULATION /

Clone I

1

Clone E 14

03lA

Con A

Culture A

cm

Culture B I

TREATED

COnA

E f Eo

f I

:

c-7

Clone m EMS

IO

E 5 I

\

Culture E f

Be- 2

ECR-I

Fig. I. Summary of the selection procedures used to isolate three independent ConA-resistant cell lines.

MATERIALS

AND

METHODS

Growth conditions and cell lines Growth conditions. Chinese hamster ovary (CHO) cells were grown as suspension or monolayer cultures in a-minimal essential medium (Flow Laboratories, Inc.) supplemented with antibiotics and 10% (vol/vol) fetal bovine serum (Reheis Chemical Co.) as reported previously [5, 61. To determine plating efficiencies [7] in the presence of ConA, culture medium containing varying concentrations of the lectin was prepared as previously described [5]. The cells gave negative tests for mycoplasma contamination as judged by plating methods [8]. The D,, value of lectin or drug for a particular cell line was defined as the concentration of agent which reduced cell survival to 10%. These values were determined from survival curves obtained when individual cell lines were plated at various cell concentrations in normal growth medium containing different doses of these agents. Cells which divided to form colonies after l&l2 days’ incubation were stained with methylene blue and -counted. These experiments were performed with cell lines that had been cultured in the absence of the lectin for at least 3 months. ConA-resistant variants. The selection procedures that were used to isolate three independent ConAresistant CHO cell lines are briefly summarized in fig. 1. CR-7 is a cloned CHO cell line isolated from an uncloned population of ConA-resistant cells (culture A) obtained by a selection method described in detail elsewhere [9].-In brief an uncloned population of ConAresistant cells was obtained after 10 passages of a nonmutagenized wild-type population at 34°C in growth medium containing 40 pg/ml ConA. CR-7is a subclone selected from the mixed population of ConA-resistant cells (culture A) and many of the properties of the uncloned population [9] and the CR-7 variant [6] have been described in detail. Exp Cell Res 114 (1978)

BCa-2 is a cloned cell line isolated from an uncloned population of ConA-resistant cells (culture B) by a selection procedure similar to the one described for the isolation of CR-7. However, culture A and culture B were derived from unique non-mutagenized clones of wild-type CHO cells [9]. Also, culture B was maintained in the presence of ConA-containing growth medium 14 times before the subclone named BCa-2 was isolated. ECa-1 was selected from a mutagenized population of CHO cells after one passage in the presence of 40 &ml ConA TlOl. A recentlv cloned nonulation of Chic cells was treated with a-mutagen’ by exposing cells growing exponentially on a 15X 100 mm tissue culture plate at 34”C, at a concentration of 1X 106cells to 300 pg/ml ethylmethanesulfonate for 17 h. The fraction survival of colony-forming ability after mutagen treatment was 0.2. The treated cells were washed with PBS, resuspended in fresh growth medium, and incubated at 34°C for 10 days to permit multiplication of the surviving cells. The cells were removed with 0.15 % trypsm solution and 1X 1Ogcells were added to another 15~ 100 mm plate containing 25 ml of growth medium wtih 40 pg/ml ConA. After-incubation-in the presence of growth medium with ConA at 34°C for 12days the plate was observed to have a single colony. The lectin-containing medium was then replaced with fresh growth medium without ConA and the cells were grown to a partial monolayer. The population was cloned [5] and named ECa-1. ConA-resistant revertant CHO cells selected for resistance to ConA cytotoxicity at 34°C are usually temperature-sensitive (ts) for growth [5, 6, 91. The ts property of the ConA-resistant line, CR-7 has been described [6], and was used to isolate a revertant of the CR-7 population. This was done by incubating 2~10~ variant cells on 16 oz. Brockway bottles (Brockway Glass Co., Inc.) containing normal growth medium at 39°C (the non-permissive temperature; [6, 93). After approx. 14 days some colonies appeared on the glass surface. A portion of the cells of one of the large colonies was removed with a sterile Pasteur pipette, added to a 15x60 mm tissue culture plate and grown to a monolayer at 39°C. These cells were maintained on 16 oz. Brockway bottles at 39°C for about 2 months and then cloned as nreviouslv described l-51. The cloned line was continuously cultured at 34’6 for 2 months and in subseauent exneriments was found to have lost the ts phenotype and to have regained a sensitivitv to the cvtotoxic effects of ConA. This cell line has been named RCa-7. Somatic cell hybrids. McBurney & Whitmore [ 1I] have isolated a CHO line autotrophic for glycine, adenosine, and thymidine; this cell line, AUXBl, was kindly provided for this study by P. Stanley and L. Siminovitch. Preliminary experiments indicated that AUXBl cells exhibited a wild-tvoe sensitivitv to ConA. An ouabain-resistant marker’was added to the auxotroohic line bv selectinn cells cauable of formine colonies in the presence of-2 mM oiabain [12]. Thg isolation of the A-7 and A-W hybrid cell lines have been described in detail [lo]. The hybrid A-W was isolated from the fusion of AUXBl (ouabain-resistant) and wild type cells (clone 1) while A-7, A-7B and A-7C were isolated from the fusion of AUXBl (ouabain-

Concanavalin resistant) cells with P-7 cells. Each hybrid line contained modal chromosome numbers/cell approximately twice the value of 21 characteristic of the P-7 variant and the wild-type cells.

Cell agglutination

assay

Cells growing exponentially in suspension culture at 34°C were collected by centrifugation, washed with phosphate-buffered saline, and suspended in 0.154 M NaCl solution at a concentration of 2x 106 cells/ml. 0.5 ml of the cell suspension and 0.5 ml of a 0.154 M NaCl solution contaming various concentrations of lectin was added to a 10x35 mm tissue culture plate. The lectin-treated suspension was slowly agitated at room temperature for 10 min and then viewed with a light microscope for cell clumping. Agglutination was scored qualitatively on a scale of - to + + + + (no agglutination to virtually complete agglutination).

Cell detachment

assay

Cells were plated at 6~ 104 cells/ml on 15x60 mm plastic culture plates and incubated at 34°C for 24 h in a CO* and humidity controlled incubator. To perform cell detachment studies [ 131the medium was removed; the cells were washed with phosphate-buffered saline and placed at 34°C in the presence of 2 ml of phosphate buffered saline containing 0.03% trypsin. At various time intervals the buffer was removed, cell aggregates were dispersed by pipetting, and the number of cells in suspension was counted with a cell counter (Coulter Electronics Co.).

ConA receptor mobility

studies

ConA receptor mobility was measured as the ability of cells to aggregate fluorescent labelled ConA into discrete aggregates or caps [ 141.Cells were added to glass coverslips at 5 x lo4 cells/cm* and incubated overnight in growth medium at 34°C. The medium was removed from plates and the cells were washed twice with phosphate-buffered saline and then incubated at 4°C for 30 min in buffer containing 60 wg/ml fluorescent ConA (Miles-Yeda). Excess label was removed and the cells were washed twice with nhosuhate-buffered saline. Capping occurred by incubating cells in warm phosohate-buffered saline at 34°C for 1 h. Coverslius were mounted directly on slides to dry without prior fixing of cells. A Zeiss fluorescent microscope was used to examine the slides. The percent of cells showing tight cap formation was routinely determined by scoring at least 200 cells of each cell type. Also experiments were repeated for each cell type at least three times.

RESULTS Sensitivity

to ConA

The cell lines described in this report were examined for the ability to form colonies in growth medium containing varying con-

A resistance

219

centrations of ConA. This was accomplished by incubating various numbers of cells at specific ConA concentrations for 10-12 days at 34”C, counting the number of colonies formed, and constructing survival curves (fig. 2a, 6). The lectin-resistant cell lines showed a similar reduced sensitivity to the cytotoxic effects of ConA (fig. 2a) CR-7, ECR-1 and BCR-2 exhibited D10values of 45, 45 and 48 respectively. The wild-type cell lines from which the three lectin-resistant variants were originally selected (fig. 1) were more sensitive than the variants to ConA cytotoxicity; a D,, value of 18 was determined for the three wild-type lines. According to the DIO evaluations approx. 2.5 times more ConA was required to reduce the relative plating efficiency to 10% in experiments with resistant cell lines when compared with experiments performed with the parental wild-type cells. P-7 cells are obviously temperaturesensitive for growth [6] and this property 100

16’

ConA cont. (@g/ml); ordinate: rel. colony-forming ability. Effects of various concentrations of ConA on the colonv-formine abilitv of wild-tvoe clone I (A); wildtype clone 2 YC); wild-type cl&e 3 (0); P-7 (A); ECR-I (0): BCR-2 (m): RP-7 (0): A-W (x): A-7 (0): A-7B (0): and A-7C (m).

Fig. 2. Abscissa:

.

II

~

I.

Exp Cell Res II4 f 1978)

220

Ceri and Wright

3. Abscissa: time (hours); ordinate: cells/tissue culture plate. Growth curves at 34°C (0); 37°C (A); and 39°C (0) on plastic tissue culture plates. In preparation for the experiments the cell lines were grown at 34°C; split into three separate cultures; one culture was kept at 34°C and the other two cultures were either shifted to 37 or 39°C.

Fig.

was used to isolate a temperature-revertant line (see Materials and Methods). These revertant cells, RCR-7, were found to be almost as sensitive to the toxic effects of the lectin as the original parental wild-type population (fig. 2b). The D,, value for these cells was about 1.4 times greater than the value found for wild-type cells. Somatic cell hybrids were formed through the fusion of two lectin-sensitive CHO cell lines, clone 1 (fig. 1) and AUXBl (ouabain-resistant) cells [lo]. It is apparent from fig. 26 that these hybrids (A-W) were almost as sensitive to the toxic action of ConA as the respective pseudodiploid wildtype cells. Also, three other hybrid cell lines (A-7, A-7B, A-7C) were independently seE,rp Cell

Rzs

114 (19781

lected through the fusion of CR-7 cells with the lectin-sensitive AUXB 1 (ouabainresistant) cell line; these hybrid lines were almost as sensitive to the presence of ConA as the A-W hybrid cells and the pseudodiploid wild-type cells. The D,,, values of ConA for A-7, A-7B and A-7C were estimated to be 1.0, 1.4 and 1.O times the value observed with either A-W hybrid cells or with pseudodiploid wild type cells. These results suggest that ConA resistance behaved as a recessive characteristic in these experiments. From the data presented in fig. 2a, b it was possible to divide the various cell lines into two very broad categories. One group was obviously resistant to ConA cytotoxicity (CR-7, ECR-1 and BCR-2) and the other group was relatively sensitive to the toxic action of the lectin (wild-type clones 1, 2 and 3, RCR-7, A-W, A-7, AJB, A-7C). To examine for a possible correlation between ConA resistance and altered growth and membrane-associated properties [6] the cellular characteristics of the resistant and sensitive cells were compared further. The results obtained with the three parental wild-type cultures (clones 1, 2 and 3) or hybrids A-7, A-7B and A-7C were found to be nearly identical; therefore, in most cases only the data obtained with one wild-type line (clone 1) and one of the hybrid lines (A-7) have been presented in detail. Growth properties The temperature-sensitive growth properties of the CR-7 population have been described in detail [6]. For example, variant cells cultured at 34°C on plastic surfaces exhibited normal doubling times of 18-20 h but were unable to significantly increase in cell number when maintained at 39°C. Fig. 3 shows the results obtained from similar growth experiments carried out with the

Fig. 4. Phase contrast photographs of (a) ECR-I cells; (b) wild-type clone 2; (c) BCR-2 cells; and (d) wildtype clone 3. X 1.50.

various resistant and sensitive cell lines described in this report (fig. 2a, b). All the cell lines grew well at 34°C. The wild-type, ECR-1 and BP-2 cell lines exhibited doubling times of 18, 24, and 22 h respectively. Also, the hybrid populations (A-W and A-7) and the revertant cells (RP-7) displayed similar doubling times of 18-22 h. In general, the growth rates of the cell lines were higher at 37°C as compared with 34°C. The doubling times ranged between 15 h (wild type) and 22 h (EP-1). It is interesting to note that the three resistant lines, CR-7 (data not shown), ECR-1 and BCR-2 actually exhibited slightly reduced doubling times at 37 as compared with 34°C.

At an incubation temperature of 39”C, the wild-type, RCR-7, A-W and A-7 cells exhibited growth rates that were very similar to the rates observed at 37°C. However, in contrast to these results the ConA-resistant lines showed a marked inability to proliferate at 39°C. The variant cells showed only a small increase in cell number within the first 24 h (BCR-2) to 40 h (ECR-1) of incubation, followed by a dramatic reduction in proliferation ability as neither the ECR-1 nor the BCR-2 cell line was able to proliferate any further at the higher temperature. These results are very similar to the previously reported observations on the growth abilities of the CR-7 cell line at 39°C [6]. ConA-sensitive cell lines grow well at in-

222

Ceri and Wright

Table 1. Cell agglutination in the presence of ConA

Lectin agglutination

Mammalian cells agglutinate in the presence of certain plant lectins and changes in cell agglutination properties usually indicate Cell line 0 50 250 500 750 1000 that important alterations to the surface Wild type - - ++ ++++ ++++ ++++ membrane have occurred [Is]. The ConACR-7 --++ +++ ++++ resistant and sensitive cell lines were tested ECR-1 - - ++ +++ ++++ IN?-7 - - ++ ++++ ++++ ++++ for agglutination properties in the presence A-W - + +++ ++++ ++++ ++++ of ConA. The results of these experiments A-7 - + +++ ++++ +t++ ++++ are shown in table 1. In agreement with BP-2 cells consistently form many small clumps of previous observations [6] higher concentracells when suspended in buffer minus lectin. Although BP-2 cells appeared to resemble P-7 and ECR-1 ceIIs tions of ConA were required to agglutinate in these studies it was not possible to estimate agglu- the resistant cells than were needed to agtination accurately. glutinate wild-type cells. Also note that the RCR-7 cell line, which has lost the lectin resistant property, exhibited an agglutinacubation temperatures between 34 and tion pattern that was indistinguishable from 39°C. ConA-resistant lines possess the the original parental wild-type line. Furtherability to grow well at 34 and 37°C but ex- more, somatic cell hybrids containing either hibit a dramatic reduction in ability to pro- two wild-type genomes or containing a lectin-resistant and a wild-type genome were liferate at 39°C. ConA-resistant cells often exhibit dis- more agglutinable than ConA-resistant cell tinctly altered cellular morphologies [6, 91. lines. The hybrid lines, A-W and A-7, were consistently found to agglutinate at slightly In agreement with previous observations, lower concentrations of lectin than pseudothe cellular appearance of ECR-1 and BCR-2 cultures were found to be quite different diploid wild-type or revertant cells; the from the morphology of the wild-type cell mechanism responsible for the increased lines from which the variants were selected agglutinability of the hybrid lines is not (fig. 4). Similar to cultures of CR-7 cells [6] understood. Clearly, ConA-resistant cells are less and unlike the wild-type line ECR-1 grew in a more disorganized or “criss-cross” pat- agglutinable in the presence of ConA than tern and large multicellular clumps or ConA-sensitive cells. In contrast to these studies have spheres were often observed at the surface observations preliminary shown that ConA-resistant cell lines exhibit of the variant cells attached to the culture plate surface (fig. 4a, 6). The appearance an enhanced agglutinability when compared of BCR-2 cultures were quite different from with sensitive lines, in the presence of varieither the wild-type or the other two lectin- ous concentrations of phytohemagglutinin resistant cultures. These variants did not (PHA) between 10 and 320 pg/ml. For exnormally form large multicellular clumps ample in the presence of 64 hg/ml PHA and the shape of the individual cells was ConA-resistant cells exhibit essentially distinctly more “fibroblast-like” when com- 100% agglutination whereas the various pared with parental wild-type cells (fig. sensitive cell lines show less than 25% clumping. These studies indicate that the 4c, d). ConA cont. @g/ml)

Exp Cell RPS I14 (1978)

Concanavalin

Table 2. Sensitivity and sodium butyrate

to phenethyl

alcohol

Cell line

Du, (phenethyl alcohol (mW

DIo (sodium butyrate) (mM)

Wild type ECa- 1 BP-2 RP-7 A-W A-7

5.1 1.8 2.0 4.6 4.2 4.0

0.74 0.35 0.33 0.70 0.70 0.68

lectin agglutination properties of the ConAresistant variants are distinctly different from the sensitive cell lines. Sensitivity agents

to membrane active

A resistance

223

butyrate that were close to the wild-type result (table 2). These studies support the view that cells selected for resistance to ConA are usually more sensitive than ConA-sensitive cells to the toxic action of certain membrane active agents. Cell detachment with trypsin

The ability of cells to adhere to a solid surface can be quantitated by measuring the rate at which the cells are removed from a solid support with a trypsin solution [13]. Similar to the findings with CR-7cells [6] the independently selected variants, ECR-1 and BCR-2 were released from the surface of a plastic culture plate at a greater rate than wild type cells (fig. 5). For example after 40 min of trypsin treatment about 40% of either the ECR-1 or BCR-2 cells were released from the culture plate, whereas, during the same treatment period only 10% of the wild type cells were released from the solid support. Studies with the RCR-7 cell line indicated that a return to ConA sensitivity is accompanied by a return to a reduced rate of cell

ConA-resistant cells are sensitive to many agents which are known to interact with the surface membrane [5, 61. The various resistant and sensitive cell lines were tested for the ability to form colonies in the presence of two of these membrane active agents, phenethyl alcohol [16, 171 and sodium butyrate [18, 191. In agreement with previous observations on the “collateral sensitivity” of ConA-resistant cells [5] the CR-7 [6], ECR-1 and BCR-2 cell lines were 50 t 0’ found to be more sensitive than the wildo40. type population to the toxic effects of these / agents (table 2). The D,, value for phen- 30. / P ethyl alcohol and sodium butyrate with wild-type cells varied between 2.0 and 2.5 times the respective values obtained with the lectin-resistant cell lines. Furthermore, the revertant cell line, RCR-7 and the two IO 20 30 40 hybrid cell lines, A-W and A-7, which ex- Fig. 5. Abscissa: time (min); or&tare: % detached hibited approximately the same plating ef- cells. Kinetics of detachment of wild type (0); ECa-1 (0); ficiency as the wild-type population in BP-2 (0); RCa-7 (A); A-W (A); and A-7 (x) cells from growth medium containing varying con- a plastic tissue culture plate with phosphate-buffered saline containing 0.03 % trypsin. Cells were incubated centrations of ConA, also showed D,,, for 24 h at 34°C in normal growth medium prior to the values for phenethyl alcohol and sodium experiment.

201/Y lops

14t-781813

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Ceri and Wright

ABCDEFG

Fig. 6. Ordinate: % cap formation. Percent cap formation on (A) wild type; (B) RCR-7; (C) A-W; (D) A-7; (E) F-7; (F) E&l; and(G) BCR-2 populations. Experiments were performed as described in the text. More than 200 cells of each celi line were examined in each of three separate experiments.

7. Distribution of labelled ConA on (a) RF7; detachment in the presence of a trypsin Fig. (b) P-7 cells after binding at 4°C followed by incubasolution. Although 60 % of the CR-7popula- tion at 34°C for 1.5 h. X700. tion was released from the surface of a culture plate during a 40 min incubation period in the presence of a 0.03 % trypsin solution fluorescent lectins. The mobility of ConA [6] only 15% of the revertant RP-7 popula- receptors at the surface of CHO cells was tion was detached from the surface after 40 visualized by the aggregation of fluorescent min of identical treatment (fig. 5). Also, the conjugated lectin into discrete caps (fig. 6). Experiments with wild-type cells indiConA-resistant property was not expressed in hybrids formed through the fusion of a cated that greater than 95 % of the cell proresistant cell with a lectin-sensitive cell (fig. duced tight caps after incubation at 34°C for 2); the A-7 detachment kinetics were very 1 h following fluorescent ConA binding. similar to the kinetics observed with Similar results were observed with each of pseudodiploid wild-type cells and A-W the other ConA-sensitive cell lines as well hybrid cells formed by the fusion of two lec- (RCR-7, A-W and A-7). Fig. 7a shows the type of cap formation normally observed tin-sensitive lines. These results indicate that the adhesive with lectin sensitive cell lines. A significant properties of ConA-resistant and sensitive difference in cap forming ability was observed when ConA-resistant cells were anacells are significantly different. lysed by this technique. Approx. 10, 15 and ConA receptor mobility 25 % of the CR-7, BCR-2 and ECR-1 populaAlterations in the surface membranes of tions exhibited an ability to form caps. Inmammalian cells may lead to significant creasing the incubation period to more than changes in the surface fluidity and receptor 3 h did not increase the proportion of varimobility characteristics [2&22]. Receptor ant cells that showed cap formation. Most mobility properties can be studied by ex- of the lectin-resistant cells showed a ranamining the ability of cells to aggregate dom distribution of label over the cell surExp Cell Res II4 11978)

Concanavalin A resistance face. Some patching was occasionally observed, especially at contact points between adjacent cells. Fig. 7b shows the distribution of label over the surface of a ConAresistant cell. Apparently the ConAresistant and sensitive cell lines have markedly different lectin-receptor mobility characteristics. DISCUSSION The plasma membrane plays an important role in cellular regulation. We have been interested in developing genetic systems for investigating cell surface regulatory mechanisms in mammalian cells [5]. Lectins like ConA are known to interact with mammalian cell surface oligosaccharide chains and to cause a variety of complex but interesting biological alterations with cultured somatic cells [4]. It seemed reasonable to expect that mammalian cells selected for resistance to the cytotoxic effects of these lectins would exhibit alterations in membrane-associated properties. The assumption has been that these variants would provide an important opportunity to investigate membrane structure-function relationships. Several laboratories have reported the isolation of cell variants which exhibit resistance to the cytotoxic effects of certain plant lectins [5,23-281. Some recent reports elegantly demonstrate the usefulness of these mutants. For example, contributions to understanding the biosynthesis and function of cell surface carbohydrates have resulted from the important studies carried out with ricin and PHA-resistant cell lines [29, 301. Mammalian cells resistant to the cytotoxic effects of ConA possess alterations to some fundamental biological properties [5, 6, 9, lo]. The complex phenotype of one of these lectin-resistant cell lines has recently

225

been described in detail [6]. However the value of this type of variant line in further investigations into membrane structurefunction relationships depends upon firmly establishing that the interesting cellular properties exhibited by the variant line are directly related to the ConA-resistant property. The results presented in this communication indicate that the required correlation exists; the ConA-resistant phenotype of three independently selected lectinresistant cell lines was shown to be directly related to modifications in specific growth and membrane-associated properties. This statement is supported by the following observations. (a) Three ConA-resistant cell lines selected from independent wild-type clones by single step (EC?-1) or cycling (CR-7 and BCR-2) methods possess similar complex phenotypes which include: obvious temperature-sensitive growth properties, altered cellular morphology on culture plates, increased sensitivity to such membrane-active agents as phenethyl alcohol and sodium butyrate, altered lectin agglutination properties, modified adhesiveness to substratum properties, and defective lectin-receptor mobility characteristics. (b) The selection of a revertant cell line (RCR-7) which exhibited a near wild-type sensitivity to ConA cytotoxicity also showed growth and membrane-associated properties that were very similar to parental wild-type cells. (c) Somatic cell hybrids formed through the fusion of wild-type and lectin-resistant cells exhibited a ConAsensitive phenotype, and possessed growth and membrane-associated characteristics that were very similar to pseudodiploid wild type cells and control cultures of pseudotetraploid hybrid cells. (d) The ConA binding properties of the various lectin-resistant and sensitive cell lines have been described recently [lo]. Important differences in the Exp Cell Res I14 (1978)

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mechanism of lectin binding and the amount of lectin bound/cell surface area were found to exist between ConA-resistant (CR-7, EC91 , BCR-2) and ConA-sensitive (wild type, RCR-7, A-W, A-7) cell lines. A direct correlation existed between the ConAresi ant phenotype and an altered mech7 anism of lectin-membrane interaction. Perhaps it should be noted that the studies reported here do not rule out the possibility of eventually isolating a distinctly different class of ConA-resistant variant which would possess another unique set of altered cellular properties since it is reasonable to expect that more than one type of cellular change (mutation or epigenetic change) may lead to a ConAresistant phenotype [9]. The specificity requirements of the interactions between ConA and saccharides indicates that the lectin binds c+rr-glucopyranosyl, a-D-mannopyranosyl and related cell surface structures [ 151. Therefore, ConA-resistant cells should contain specific changes in cell surface carbohydrate-containing structures. Some recent experiments in our laboratory with several surface labelling techniques have indicated that ConA-resistant cell lines contain specific alterations in cell surface glycoproteins that are not found with various ConA-sensitive cell lines [31-331. A major difference between resistant and sensitive cell lines is the presence on resistant lines of a high molecular weight glycoprotein (mol. wt 155000) which is missing from the surfaces of sensitive cell lines. There is also a good indication that ConA-resistant cells may be defective in the ability to synthesize an oligosaccharide-lipid intermediate which is needed for the addition of mannose to proteins ([28], Wright, Ceri and Jamieson, unpublished observations). Also, we have observed changes in several other enzyme acExr, Cell Res I 14 ( 1978)

tivities associated with the biosynthesis of surface glycoproteins in the lectin-resistant cell lines; this work is still in progress and will be published at a later date (Blaschuk, Wright & Jamieson, unpublished results). Therefore, we are just beginning to understand the molecular basis for the complex phenotype associated with ConA resistance. It is clear that the variants provide a novel opportunity for investigations into membrane structure-function relationships. We thank the NRC of Canada for a research grant (J. A. W.) and a scholarship (H. C.). We are grateful to Drs L. Van Caeseele and A. Olchowecki for advice concerning photomicroscopy techniques. Also, we thank Miss S. Korouatnick and Mr C. Parfett for aid at various stages of the project.

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