Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of...

[N VITRO V.I. 12, No. 3, 1976

S E L E C T I O N AND CHARACTERIZATION OF CHINESE HAMSTER O V A R Y C E L L S R E S I S T A N T T O T H E C Y T O T O X I C I T Y

O F L E C T I N S 1

PAMELA STANLEY AND LOUIS SIMINOVITCH

Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada MSS IA8

S U M M A R Y

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal a-mannose residues is dramatically reduced (~<5% of the activity detected in wild-type CHO cells) in several independent Pha R clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind ~2sI-Con A; and (e) decreased surface galactose residues--all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4 -5 "lectin-resistance" phenotypes have been demonstrated. Complementa- tion data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.

Key words: somatic cell genetics; lectins; membrane mutants; glycosyl transferases; complementation.

There has been increasing evidence in recent years that cell membranes play a multiplicity of complex roles in cell function. Among the several approaches that can be made to the elucidation of these roles, genetic analysis offers some particular advantages. It can be expected that a variety of structural and functional properties of membranes will be altered by mutation. At the same time each particular genetic event will lead to a specific modification which can then be studied comparatively in isogenic cell lines. The strength and precision of the genetic approach to studies of over-all cell function and regulation,

1 Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2-5, 1975.

including that of cell membranes, has already been demonstrated clearly with prokaryotes. For these reasons, our laboratory initiated a program a few years ago designed to exploit genetic methods for the study of membrane structure and function. As might be expected, mutants involving a variety of membrane alterations have already been found. Ouabain-resistant mutants are altered in the Na/K activated ATPase situ- ated in the membrane (1), and colchicine- resistant mutants are altered in their permeability to colchicine and a variety of other drugs (2).

In our own studies, we have been interested in membrane alterations engendered by mutation to resistance to the cytotoxicity of plant lectins. In addition to their known properties as mitogens and agglutinating agents, lectins are also toxic

208

LECTIN-RESISTANT CHO CELLS 209

to some types of cells cultivated in vitro (3, 4). There is evidence that lectins exert their mito- genic and agglutinating action by binding to exposed carbohydrate residues of the cell membrane. Although in many cases the mechanism of cytotoxicity is unknown, binding at the cell membrane is probably a necessary first step in this process. Therefore it seemed reason- able to expect that, of mutants selected for their resistance to lectin cytotoxicity, some would show decreased binding of the lectin, which might reflect important modifications in the structure and/or conformation of the membrane.

The cell line we have chosen for our studies is tile Chinese hamster ovary line (CHO) originally isolated by Puck and co-workers (5). This line has several advantages for genetic study: ease of manipulation; ability to grow in suspension culture; a relatively stable karyotype; a high plating efficiency; and the existence of several auxotrophic CHO lines (6). The feasibility of selecting lectin-resistant CHO lines was originally demonstrated by Wright (7), who isolated and studied concanavalin A-resistant cells.

In our laboratory, we have studied cells resistant to the phytohemagglutinin from Phaseolus vulgaris (PHA), and subsequently a variety of other lectin-resistant (Lee ~) cells. Our approach has been to isolate Lee R cell lines, and then to examine their genetic, physiologic and biochemical properties. We have chosen two auxotrophs as parental l i nes - -one which requires proline for growth (Pro-) and a second one which requires glycine, adenosine and thymidine but is reverted for the proline requirement (Gat Pro+). Since a great deal of the detailed work describ- ing these studies has either been published (8, 9), or is in press (10), or in preparation, in this paper we present mainly an overview, including only experimental data which is not to appear elsewhere.

Isolation of PHA-resistant (Pha R) cells. In the presence of 10/xg/ml PHA or higher, about 1 to 2 in 105 CHO cells survive the cytotoxic action of the drug (8; Table 1). The number of resistant colonies seems to be greater in cells derived from older clones (Table 1). All cells that survive low concentrations of the lectin are also resistant to concentrations of PHA up to 1 to 2 mg/ml, indicating that a high degree of resistance is acquired in a single step (8). More than 100 such PHA-resistant clones have been isolated either

TABLE 1

FREQUENCY OF P H A R P H E N O T Y P E IN

D I PL O I D C H O CELLS

Time Cultured before Pha R Colonies Exposure to PHA

Cell Line (per l0 G viable cells) (months)

Pro- 0 4 1.5 Pro- 0 30 2.5

Pro 1 5 0.5 Pro- 2 2 0.5 Pro- 3 3 0.5

Gat-Pro ÷ 0 10 1.3 Gat-Pro + 0 15 2.0

Gat-Pro + 1 5 0.5 Gat-Pro + 2 5 0.5 Gat -Pro + 3 5 0.5 Gat Pro + 4 5 0.5

Different CHO clones were plated in the presence of 10-25 /xg/ml PHA and the survivors were scored. Most of the colonies that arose were picked and shown to retain their PHA-resistance after subsequent cultur- ing in nonselective media.

with or without mutagenesis, and at least 98% of them remain resistant on subsequent culture in the absence of the selecting agent. In one study, cells retained their Pha ~ phenotype after being continuously cultured for 300 generations in the absence of PHA (8). The karyotype of Pha R cells is essentially identical to that of the parental line (8).

Genetic characterization of Pha R cells. As indicated above, Pha R cells breed true. Further genetic characterization of the system has been made by measuring the rate of mutation to PHA- resistance using the Luria-Delbruck Fluctuation Test (11), by examining the effects of a mutagen, and by determining the behaviour of the marker in somatic cell hybrids.

The results of the Luria-Delbruck Fluctuation Test are shown in Table 2. Two conclusions can be derived from this data. First, the rate of generation of Pha R cells is nonrandom and is therefore compatible with a mutation event. Secondly, the observed rate of mutation, using the Median method of calculation (12), is 1.5 + 0.3 × 10 -s per cell per generation. The frequency with which Pha R cells can be isolated in hybrids formed between the two parental auxotrophs is of the order of 1 in 2 to 4 × 106 viable cells. This is to be compared with the frequency in quasi-diploid cells of 1 to 2 in l0 s

210 STANLEY AND SIMINOVITCH

TABLE 2

RATE OF MUTATION OF DIPLOID CHO CELLS TO PHA-RESISTANCE

(Fluctuation Analysis)

Test Control

No. replicate cultures 21 1 No. sampiings per

culture 1 25 Initial No. cells per

replicate 100 - - Final No. cells plated 3.6 -+ 0.6 × 10 e 1.2 × 10 e No. of replicates with N

Pha n colonies: N = 0 0 0

1-4 1 0 5-8 4 0 9-16 6 0 17-32 4 15 33-64 3 10 65-128 1 0 129-256 1 0 257-512 1 0

No. Pha R colonies per replicate:

Range 1-319 21-43 Median 16 32 Mean 41 31 Variance

Ratio 120 0.74 Mean

Mutation rate (Median Method) 1.5 +- 0.3 × 10 -s per cell per generation

Replicate suspension cultures of Pro- 5 cells were initiated at 100 cells per 11 ml and allowed to grow to approximately 3-4 x 105 cells per ml. The cells were counted from a sample of 0.1 ml and all the cells from cultures containing 3-4 × 105 cells per ml were plated at 1.2-1.4 × 106 cells per plate in 20 ttg/ml PHA. As a control, 25 replicate plates containing 1.2 × l0 s Pro- 5 cells (from a maintenance culture) in 20 gg/ml PHA were set up. After 8 days at 37 ° C independent colonies from a number of test plates were picked into medium containing PHA and all the plates were stained. Colonies containing >50 cells were scored and the results analyzed by the median method of Lea and Coulson (12). Seventeen of the 23 colonies picked from test plates had grown to 3-5 × l0 s cells after 12 to 18 days in medium containing PHA (10 ttg/ml). The remainder may have required passage in monolayer culture before adapting to growth in suspension (8).

cells. Thus the frequency of isolation of mutants in quasi-tetraploid cells is not compatible with a model involving two independent mutational events.

The spontaneous frequency of mutation to PHA resistance can be increased by mutagenesis with ethylmethane sulfonate (EMS) (8). Although

the system has not been examined in detail by (for example) varying the expression times, the extent of the increase in several experiments varied from about 5- to 150-fold.

It was of interest to determine whether the Lec R marker behaved recessively or dominantly in somatic cell hybrids. Hybrids were thus con- structed between parental and Pha R cells using the complementary Pro- and Gat- auxotrophic markers for selection of the hybrid cells. Such cells were found to be sensitive to PHA, indicating that Pha R behaves as a recessive trait (8).

In summary, cells resistant to PHA behave as authentic somatic cell mutants. They can be selected in a single step; they are stable in the absence of the selective agent; they occur ran- domly; their frequency is increased by mutagenesis; and they behave recessively in somatic cell hybrids.

Properties of Pha R cells. The potential useful- ness of Pha R (and other Lec R cells) depends on the identification of the biochemical and functional alteration(s) that have occurred in these

cells. One obvious possible result of a genetic change would be the loss of membrane receptors, thus inhibiting binding of the lectin to cell surface glycoproteins. A consequence of such a lesion would be a specific decrease in the available surface carbohydrate (and possibly protein) in Pha s compared to parental cells. To this end we have investigated the ability of Pha a cells to bind iodinated lectins at the cell surface (8), the availability of exposed ultimate and penultimate galactose residues, and the labeling of intact cells via lactoperoxidase-induced iodination (9).

Comparison of the binding of [125I]-PHA to parental and Pha R cells has shown that the mutants are indeed defective in their ability to bind PHA. The extent of this decrease varies somewhat from one mutant to another and does not seem to be directly correlated with the degree of resistance to the lectin. The availability of surface galactose residues was shown to be markedly decreased in Pha a compared with parental cells by comparing the gel patterns of galactose:3H-borohydride labeling of neuraminidase-treated intact cells. On the other hand, the basic pattern of membrane components iodinated via lactoperoxidase did not differ markedly between Pha R and parental cells. However there was a shift to lower molecular weight of a signifi- cant proportion of the iodinated species.


The specificity of lectins for binding glycoproteins residues partly in their simple sugar specificities (3, 4). Therefore the possibility that mutation to PHA-resistance may alter the sensitivity of Pha R cells to lectins of different specificities was examined (8, 10). The results showed that Pha R cells with the binding and surface-labeling properties described above are also highly resistant to wheat germ agglutinin (WGA), the toxin from Ricinus communis (RIC), and the agglutinin from Lens culinaris (LCA). However, they are 4- to 5-fold more sensitive than parental cells to the cytotoxicity of concanavalin A (Con A). These lectin sensitivities are reflected qualitatively in the leetin-binding properties of Pha" cells. That is, Pha R cells have a decreased ability to bind iodinated WGA, LCA and RIC whereas they bind two to four times as much [12q]-Con A as parental cells.

The binding of lectins to cell surfaces is not strictly governed by their simple sugar specificities. In fact, the evidence obtained with an isolated oligosaccharide from erythrocyte membranes (13), from competitive binding studies of sequentially-degraded porcine thyroglobulin with intact lymphocytes (14), and from the interaction of different lectins with immunoglobulin glycopeptides (15, 16) suggests that a numt)er of lectins bind to the same oligosac'charide "receptor" but at different sites along the carbohydrate chain. Thus if PHA binds to CHO cells in similar fashion, the lectin-resistance and lectin-binding phenotypes of the Pha 1¢ cells described above are consistent with an alteration in the carlm- hydrate structure of the membrane PHA binding sitels). Evidence for this hypothesis has been obtained in an investigation of the glycosyl transferase activities of extracts from Pha j¢ ceils (10). [t was found that Pha R cells possessed very low activity for the transfer of N-acetylglucosamine (GleNAe) in fl-linkage to the exposed o~- mannosyl residues of ~-galactosidase, hexosaminidase-treated lg(; glycopeptide (a gift from S. Kornfeld; 15). In eontrasl, the Pha j~ cell extracts possessed approximately 50% of wild- type (WT) activity for the transfer of GlcNAc to terminal o~-mannosyl residues of desialized, degalactosized, hexosaminidase-treated a~-acid glycoprotein (a gilt from H. Schachter: 17), and approximately 75% of the activity of parental cells tbr the transfer of GIcNAe to untreated RNase B. This variation in activity with different exogenous aeeeptors suggests that these

Pha R cells lack one of two or more GlcNAc transferases present in WT CHO ce l l s - - t he one specific for glycosidase-treated IgG as an exogenous acceptor. Appropriate controls have shown that it is unlikely that the inability to detect this GlcNAc transferase in these Pha R cells is due to an increased glycosidase or nucleotidase activity or that the pH and cation requirements of the enzyme have merely been altered.

Possible molecular basis for the origin of Pha n CHO cells. The possibility that the GIcNAc transferase lesion is the molecular basis for the origin of Pha R CHO cells is certainly consistent with their known phenotype. Thus the absence of a GIcNAc transferase activity that adds GIcNAc to a-mannosyl residues in certain carbohydrate chains would preclude the subsequent addition of galactose and sialic acid even though the en- zymes for these transfer reactions appear to be present in Pha R cells (10). The high degree of resistance of Pha" ceils to PHA, WGA, RIC and LCA and their decreased ability to bind these lee, tins may be explained by the absence of terminal sialic acid --~ galactose ~ GlcNAc se- quences on many membrane glycoproteins. The consequent increased exposure of normally in- ternal mannosyl residues probably explains the increased sensitivity and binding of Con A to Pha R cells. Finally, the markedly decreased ability of neuraminidase-treated Pha R cells to be labeled via galactose oxidase and :~H-borohydride may be explained by a lack of penultimate galactose residues in incompleted membrane glyeoproteins; and the shift to lower molecular weight of many of the surface membrane species iodinated via lactoperoxidase is consistent with decreased glycosylation of exposed glycoproteins. Pha I~ cells possessing this complex phenotype will henceforth be referred to as Pha R Type 1 (Pha%.

Selection of other Lec B mutants of CHO cells. The results described above provide strong in- dications that a specific membrane alteration can be identified with mutation to PHA resistance. This, plus the fact that resistance to PHA in Pha", CHO cells leads to an alteration in their sensitivity to a number of other lectins, sug- gested that a comprehensive analysis of membrane aherations engendered by mutation to leetin resistance could be obtained by isolating a family of lectin-resistant cells. We therefore have selected mutants resistant to WGA, RIC and


LCA. In addition, we had available to us mutants resistant to concanavalin A (Con A), supplied by R. M. Baker. The phenotype of most of these lines have not, as yet, been examined with the detail descr ibed earlier for Pha R lines. How- ever, on the basis of the known alteration in a specific glycosyl transferase activity for this lat ter Lec R line, the consequent alterations in the glycosylation of membrane glycoproteins, and the cross-resis tance patterns of Pha R' to other lectins, it might be expected that Lec R cells, selected with different lectins, would also show alterations in sensitivity to other lectins. Thus we have compared the cross sensitivity of each Lec R line to

five lectins, using a parameter called the D~0 value, which is defined as the dose of drug which reduces the relative survival of the cells to 10%

(Table 3). Several facets of this data are of interest. It is

apparent that more than one Pha R phenotype may be selected from CHO cells. In fact, four of the five lectins give rise to more than one Lec R

phenotype, based on their lect in-resis tance patterns. This is probably only a fraction of the phenotypes potentially selectable with these five lectins, since the data in Table 3 was obtained in a limited sample. The exis tence of more than one mechanism of resis tance to a given lectin is to be expected from the model (described earlier) for Pha R'. For example, res is tance to PHA may arise due to the loss of a galactosyl t ransferase enzyme, rather than a GlcNAc transferase. Thus Pha R,, cell exhibit WT sensitivity to Con A and to the other three lectins. Also, these cells bind amounts of [1~5I] Con A similar to WT cells, although they still bind low amounts of [125I]-PHA. In addition, Pha R" does not show any loss of the GleNAc transferase, when des ia l ized , dega lac tos ized , hexosamin ida se - treated al-acid glycoprotein is used as an

exogenous aeeep to r (10). Thus the Pha ~. mutant differs from Pha R' in respect to its cross-sensit ivity patterns, its binding character is- tics, and enzyme content in such a way as to

TABLE 3

LECTIN-RESISTANCE PHENOTYPES IN CHO CELLS

Lec a a Phenotype Selective Phenotype GIcNAc

Lectin Name PHA Con A WGA RIC LCA Transferase

PHA Pha R' R S R R R - WGA Wga R' R S R R R - RIC Ric a' R S R R R - LCA Lca a' R S R R R - WGA Wga R'' S WT R S S + PHA Pha R'' R WT WT WT WT + RIC Rie R" S WT S R WT + Con A Con A "~h R R R R R + PHA then Con A Pha R' Con A R''b R R R R R - WGA then PHA Wga R'' Pha R R S R S R - WGA then RIC Wga R'' Ric a R S R WT S +

A summary of the different Lec a phenotypes selected from Pro- and/or Gat-Pro + CHO cells (Stanley and co-workers; manuscript in preparation). The cytotoxicity of PHA, Con A, WGA, RIC and LCA for each of the isolates has been determined from survival curves performed with each lectin. The dose of lectin giving 10% survival (D10) for each Lec R line has been compared with the D10 for that lectin for WT cells.

a R = resistant by >~2-fold than parental cells S = sensitive by ~>2-fold than parental cells

WT = essentially identical to parental cells

b Although superficially the two Con A R lines appear to have similar Lec R phenotypes, the degree of resistance of the Pha R' Con A a'' ceils to each lectin is much greater than that exhibited by the Con A R~ cells.

The activities of the GlcNAc transferase(s) in cell extracts prepared from each of these lines have been obtained using desialized, degalactosized, hexosaminidase-treated c~-acid glycoprotein as an exogenous acceptor. + = full activity compared with parental cells; - = -50% activity consistent with the loss of the GlcNAc transferase which is specific for glycosidase-treated IgG as an exogenous acceptor. Only a summary of these results is presented here since the data are being prepared for publication elsewhere. The glycosidase-treated c~-acid glycoprotein was a gift from Dr. H. Schachter, University of Toronto.


suggest that the phenotype may have arisen due to the loss of a galactosyl transfcrase activity.

One characteristic of many of the Lec r~ lines is their acquisition of increased sensitivity to other lectins. This feature of lectin resistance is of interest in at least two ways, since it provides the potential opportunity of obtaining either revertant or multiply-marked lines. It ap- pears that the second event is by far the most frequent. PhaR,cells are Con A-sensitive and do give rise to surviving colonies when subjected to selection with Con A. However this "resistance" is often not maintained on subculture, indicating there is very low fidelity in this selection system (Table 4). Among the few survivors which do breed true, there appear to be no revertants, and these Con A a cells are still PHA-resistant (Table 4). In fact, they have been shown still to lack the GlcNAc transferase activity characteristically absent in Pha R, cells.

Similarly, isolation of survivors to PHA or RIC from Wga a,' cells does not give rise to revertants (Table 3). It should be stressed that these results do not necessarily mean that reversion cannot occur for these loci, since the frequency of such reversion may be low and, consequently, may be difficult to detect due to the occur- rence of alternative events. The significance of these observations will be discussed further below.

Among the primary isolates, it seems most probable that Pha a,, Wga a,, Ric R, and Lca R' represent similar basic lesions. Their lectin- resistance patterns are essentially identical and

they all lack the specific GlcNAc transferase activity missing in Pha R, cells (Table 3). Conversely, at least five distinctive Lec R phenotypes can be distinguished in primary isolates. It will be noted as well that the phenotypes of double mutants selected by consecutive isolation, such as Phaa, Con A R, are further modi- fied, as indicated by their lectin-resistance patterns. There is strong evidence that the Con A R marker in the double mutant differs from the Con A R marker in cells isolated in a single step. First, primary Con A R isolates are temperature-sensitive for growth, whereas PhaR,Con A R cells are not. Secondly, the two Con A R phenotypes appear to complement each other in somatic cell hybrids (Table 5). Thus there are potentially eight distinct phenotypes that can be isolated by these methods.

DISCUSSION

Lectin-resistant cells are easily selected from quasi-diploid CHO cell populations. The find- ing that these markers behave recessively in somatic cell hybrids indicates that each may be present in only one copy in parental cells. We have not yet demonstrated that resistant populations arise by point mutation, although the several genetic parameters that have been examined are consistent with this interpreta- tion. However the failure to isolate revertants under appropriate selective conditions indicates that perhaps other genetic mechanisms might be involved. For example, loss or rearrangement of the chromosome carrying the relevant gene

TABLE 4

CON a SELECTION OF PHA R CELLS

Experi- Tempera- Frequency of Colonies No. Con A R No. Pha" Cell Line ment ture (per 10 ~ viable cells) No. Tested No. Tested

Gat-Pro + 2 PhaR'I 1 37 ° 2-3 0/4 N.D. b 2 37 ° 2--3 0/3 N.D. h 3 34 ° 2-3 2/4 4/4 4 34 ° 40- < 1 0/87 87/87

Pro- 5 PhaRq-I 1 34 ° 1-2 2/58 a 58/58 2 39.5 ° 13 0/24 24/24

Pha R CHO cells from two independent clones were subjected to selection in 7.5-15/zg/ml Con A in a variety of experiments. Many of the colonies that arose were tested after subculture for their resistance to the selective dose of Con A and to various doses of PHA.

a These isolates were found to be pseudotetraploid. b N.D. = Not Determined.


TABLE 5

COMPLEMENTATION BETWEEN CON A R CHO CELLS

Lec R Phenotypes Comple-

Phenotypic Crosses PHA Con A mentation

PhaR'Con A R'' × WT WT WT + PhaR'Con A R" × Con A a' WT WT + Phaa'Con A R't × Pha R' R S -

A summary of the Lee a phenotypes of somatic cell hybrids formed between different Con A R phenotypes (Stanley and co-workers; manuscript in preparation). Cells of the appropriate Lec R phenotypes and containing complementary auxotrophic markers were grown in mixed culture, treated with inactivated Sendai virus, and hybrids were selected in medium that lacked the auxotrophic requirements as described previously (8). Selected hybrids shown to be pseudotetraploid were cloned and their D10 values for PHA and Con A were compared with those of the parental lines.

could also give rise to the Lec R phenotype. Evidence on this point will have to await biochemical and immunological characterization of the "missing" GIcNAc transferase activity from Pha R cells. Alternatively, we are attempting to isolate cells of temperature-sensitive Lec R lines. The mere existence of such lines would, of course, provide evidence for the presence of an altered gene product in the cell. Moreover, temperature- sensitive Lec R lines would allow for easy selection of revertants.

The frequency of appearance of Pha R cells in quasi-diploid and quasi-tetraploid cells is of interest in respect to the nature of genetic variation in somatic cells. As indicated in our re- suits, the frequency of Pha R mutants in the former class of cells is of the order of 10 -~, whereas in the latter it is about 10 -6 , rather than the expected 10 -l° for a recessive mutation. This type of result has been obtained by several investigators for other markers (18, 19; Harris and Whitmore, unpublished observations); the most likely explanation being that in quasi- tetraploid cells the resistant phenotype is gener- ated by both mutation and segregation, whereas in quasi-diploid cells, the major basis of the genetic variation is mutation.

It will be of interest to determine the number of complementation groups concerned with resistance to the five lectins that we have examined so far. In addition, the fact that Lec R cells are sensitive to other lectins allows one to isolate lines carrying several Lec a markers. With such lines, it should be possible to con-

struct appropriate hybrids to determine the syn- teny relationships of the individual leetins.

Leetin-resistant animal cells have now been isolated in a number of laboratories (20-28). Many of these isolates have been characterized only with respect to their growth properties and the level of their resistance to the selective lectin. More recently, however, Ricin-resistant cells that possess a decreased activity for GlcNAc transferase as well as a complex leetin- binding phenotype have been reported (25, 27, 28; Table 3). The Lee R phenotype exhibiting a decreased GlcNAc transferse activity is certainly the best-characterized and may be the most common since it probably can be selected with any of at least four lectins (Table 3). However Lee R phenotypes that do not possess markedly decreased binding for the selective leetin nor an altered GlcNAe transferase have also been isolated (26, 28; Table 3). These phenotypes also occur with relatively high frequency. Thus probably only a fraction of the possible Lee R phenotypes have so far been described. For example, Lee R cells that are not altered in their ability to bind the selective lectin are presum- ably resistant to its toxic effects via another mechanism.

The fact that a mutation in a specific enzyme (GIcNAc transferase) could account for at least one Lee R phenotype, and that many other Lee R phenotypes which may arise via similar mechanisms exist, suggests that Lec R animal cells will prove to be a powerful tool in the investigation of membrane structure and function, and in the elucidation of the biochemistry of glycoprotein biosynthesis in animal cells. Specific membrane mutants of mammalian cells also should be very useful in virology and in the study of the immunology of cell surfaces.

Note added in proof." The data summarized in Tables 3 and 5 has recently been published in detail (Stanley, P., V. Caillibot, and L. Simino- vitch, 1975. Selection and characterization of eight phenotypically distinct lines of lectin-resistant Chinese Hamster Ovary Cells. Cell 6: 121-128.)

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A C K N O W L E D G M E N T S

The authors wish to thank V. Caillibot, Wendy MacDougall and Nancy Stokoe for excellent technical assistance.

The research was supported by the Medical Research Council of Canada (MT-4732), National Cancer Insti tute of Canada and the National Inst i tutes of Heal th of the United States.

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