In, J Biwhem. Vol IO. pp. 545 Lo 549 0 Prrpamon Press Ltd 1979. Punted IO Great Britam
CHANGES IN CELL SURFACE ON VARIANT HAMSTER
GLYCOPROTEINS CELL LINES
H. CERI and J. A. WRIGHT Department
of Microbiology,
University (Receiced
of Manitoba,
15 Nocemher
Winnipeg.
Manitoba,
Canada
1978)
Resistance to concanavalin A was accompanied by modifications to the fundamental growth and membrane-associated properties of two concanavalin A-resistant cell lines which were independently selected by either single step or cycling procedures. 2. Using the galactose-oxidase [‘H]borohydride surface labelling procedure, several modifications in surface glycoproteins were detected on variant cells when compared to parental wild type cells; a prominent difference was the presence of an additional surface component, with an apparent molecular weight of N55,OOO on variant cells which was missing from both wild type populations. 3. These observations provide strong support for the view that modified cell surface glycoproteins are directly involved in the complex concanavalin A-resistant phenotype which includes changes to some fundamental biological properties; concanavalin A-resistant variants provide an opportunity to correlate changes in membrane function with modifications in membrane structure.
Abstract-l.
INTRODUCTION
plasma membrane of mammalian cells plays a key role in the regulation of a variety of cellular events. A great deal of evidence supports the view that the cellular membrane is intimately involved in such basic biological phenomena as cell growth, the immune response, intercellular communication, and cellular differentiation (Quinn, 1976; Nicholson, 1976). Also many biochemical differences at the cell surface have been found between normal and transformed cells; these differences appear to be important for expressing the potentially malignant properties of the transformed cell (Burridge, 1976). However much more information concerning relationships between membrane structure and function is required before a proper understanding of some of the complex regulatory mechanisms associated with the surface membrane is achieved. Recent advances in the field of somatic cell genetics have provided new approaches for investigating membrane properties (Till er al., 1973; Siminovitch, 1976). Correlations of functional changes with membrane structural alterations resulting from mutations can serve to clarify obvious, as well as subtle, details of structure-function relationships (Till er al., 1973). With this view in mind we isolated and characterized Chinese hamster ovary (CHO) cells which exhibit reslstance to the cytotoxic effects of the lectin, concanavalin A (Wright, 1973). This protein is being used as a tool to investigate many aspects of membrane biology (Brittiger & Schebli, 1976) since it interacts with specific oligosaccharide chains at the surface membrane (Sharon & Lis, 1972). Independently isolated concanavalin A-resistant cell lines generally exhibit a very complex phenotype which includes modifications in specific growth and membrane-associated properties. For example these lectin-resistant variants exhibit obvious temperature-sensitive growth properties, altered cellular morphology, increased sensitivity to some membrane-active agents, altered lectin aggluThe
545
tination properties, modified adhesiveness to substratum properties, altered lectin binding characteristics, and defective lectin-receptor mobility properties (e.g. Wright, 1973; Wright, 1975; Ceri & Wright, 1977a,b; Wright & Ceri, 1977; Ceri & Wright, 1978 a,b). Clearly, it is important to identify the surface modifications involved in concanavalin A resistance since it will lead to a better understanding of some fundamental principles of membrane function (Till et al., 1973). Recently, we used specific cell surface labelling techniques to examine the surface of a concanavalin A-resistant cell line in detail (Ceri & Wright, 1978a). The variant cells exhibited several changes in surface labelling patterns when compared to parenta! wild type cells: a major difference was the presence of an additional surface component, with an apparent molecular weight of 155,000 on resistant cells which was missing from wild type cells. When the galactose-oxidase-(‘H)borohydride technique was used the extra component accounted for 12-18”/, of the total labelled cell surface glycoprotein on resistant cells. Lactoperoxidase catalyzed iodination of surface polypeptides and the metabolic incorporation of labelled glucosamine into membranes of resistant and sensitive cells also revealed the presence of a high molecular weight surface structure on the concanavalin A-resistant but not sensitive cells. Therefore it seems that concanavalin A resistance is accompanied by alterations in cell surface glycoproteins, and that these changes are directly involved in determining the complex biological changes previously described for concanavalin A resistance (Ceri &Wright, 1978a). However, since much of the data presented to support this contention comes from a detailed study of the membrane glycoprotein content of a single concanavalin A resistant cell line, it is necessary to carry out further studies on the relationship between the complex concanavalin A resistant phenotype and cell surface glycoproteins with other independently selected resistant
H. CI KI and J. A. WRIGHT
546
and sensitive cell lines. As previously Wright. lated
197&t) variants
further are
very
establish
this interesting
describe
the results
studies
with
important correlation;
of such
a study
noted
(Ceri
independently
in order
to firmly
in this report and
& iso-
provide
we evi-
which strongly supports the idea that there is a direct relationship between the fundamental biological changes and an obvious alteration in surface
dence
glycoprotein
content.
\IATERI.ZI.S
A[\iD
METHODS
Chinese hamster ovary (CHO) cells were grown as suspension or monolayer cultures in cc-minimal essential medium at 34 (Flow Laboratories. Inc) supplemented with antibiotics and lo”,, (rol vol) fetal bovine serum (GIBCO Ltd) as described previously (Wright. 1973: Cerl & Wright. IY77a.b). Plating efiiciencies were determined by standard techniclues (Wright. lY73).
A concana\alin A-resIstant cell line was selected from a CHO population 1%hich was previously mutagenized with ethyl methane zulfonate (Eastman Kodak). Ceils (I x IO”) growing exponentlall) on ii IS x 100 mm culture plate at 34 were Ireatcd with 350 11 ml of the mutagen in growth medium ior 15 hr. The treated cells were washed with phosphate-bulfrrcd haline. resuspended in fresh growth medium without mutagen at 34 for 2 weeks 10 allow sur\ icing cell< to grow.. These cells were then subjected to ;I \ingle step-selectIon procedure by adding 1 x lOh mutaeenized cells to a I6 oz Brockway bottle (Brockway Glass ?o.) contalnlng 30 ml growth medium supplemented with 4Ojrg ml concana\alin A (Calbiochem. Co.). Note: since concanavalin A interacts with serum components in the 1973) the lectin-containing growth medium (Wright. medium has routinely Incubated at 34 for 24 h and the resulting precipitate was removed either by centrifugation or (iltrntlon: medium containing concanabalin A was always cleared of precipitate before being used In the selection of variant cells. After approximately t*o weeks in the presence of lrct~n the sur\i+ing cells were removed and the culture was cloned as described previously (Wright. 1~73). One clone was selected for further study and was referred to as \arlan-I (V-l): these cells here routine11 cultured in medium lacking concanavalin A 4 second ~arlant cell line wa:, selected from a non-mutageniLed wild type CHO population b! a cycling procedure (Wrlphr. lY73: Wright. lY75). Approximately I x IOh cells were added lo a lhor Brockua) bottle containing 30ml growth medium with 40~1.g ml concanavalin A and incubated al 34 for about 5 days. After treatment with lectin I Iable cell\ \\ere allowed 10 recover in the presence of normal growth medium lacking concana\alin A. After 7 IO days about I x IO” cells were again placed m a 160~ BI-ockwaq bottle contaming medium supplemented with 40 ,I@ ml concann\aI~n A. This procedure of exposing cells lo lectin followed by a recovery period was performed four times. Then from this point on cells were continuously exposed lo medium with concanavalin A: every time a monolayer formed the cells were removed and I x IOh cells were added to medium containins 40/l@ ml lectin. The population was exposed to concanavalin A-containing medium a total of I4 times. and was then cloned as prepiously described (Wright. 1973). A clone was selected and referred lo as \arlant-2 (V-2): these cells were cultured in medium lacking concanavalin A. After approximately 6 months of continuous culture in the absence of a selectee agent the two variant cell lines nere used 111the esperlmentc described in this report.
Cell surface labelling of galactose and galactosamlnc residues on cells previously grown in logarithmic suspcnsion culture was performed by the galactoxe oxidase-[2H]borohydride method (Cerl & Wright. 1978a; Cahmhcrg & Hakomori. 1973). Protein extraction and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoreais M;I\ carried out by the method of Laemmli (Laemmli. lY70). Gels were sliced into approximately I.5 mm scct~on\. digested in H,OZ, and counted in a toluene-based cocktarl with a scmtillation counter. Molecular weights were determined by comparing the distance a particular peak mo\ed in a gel with the distance that known molecular wcelght markers migrated In the gel (Cerl & Wright, 1977b: (‘en & Wright, 1978a). The markers that were used included serum albumin (69.000). ovalbumin (44.500). myoglohm (17.800). and cytochrome (’ (12.400). Also included \\cre the E. w/i RNA polymerase B’. B. n and r subunlts (gencrously provided by Dr C T. Chow) with molecular wclghtq of 165.000. 155.000. 95.000 and 39.000 respect iveIl.
Most biochemicals were purchased from the Sigma Chemical Co. and radio chemicals were obtained from New England Nuclear
RESULTS
The eRects of various concentrations of concana\aefficiency of the wild type and the lectin-resistant cell lines. V-l and V-2, were determined by incubating various numbers of cells for IO days at 34 in the presence of different concentrations of the lectin. and counting the number of colonies formed (Wright, 1973: Ceri & Wright. 1978b). It is obvious from Figs 1 and 2 that the concanavalin A-resistant lines are significantly more successful than the parental wild type populations at forming colonies in the presence of the lectin. For example. in the presence of 18pg! ml concanavalin A the wild tj pe populations showed a relative plating etficiency of lin A on the plating
IO 20
30 C 0
40 N
50 A
102030405 &q/ml
Fie. I. EtTec1 of Lariouc concentrations of concana\ 31111,\ on the plating efficiency of mild tlpc cell line\ in !hc absence and presence of the hapten. IO ’ M mcth>l Y-I)glucose. Wild type line from which V-l was selected. In the absence (0) and presence of hapten (0). Wild type line from which V-l was selected. in the absence (A) and pre\ence of hapten IA). Fig. 2. Effect of Larious concentrations of concana\.1/1n A on the plating efficiency of lectin-resistant cell line\ in rhe absence and presence of the hapten. IO-’ M methyl r-o-glucose. V-l in the absence (A) and presence (Cl ~lf hapten. V-2 in the absence (0) and presence I@) of hapt
CHANGES IN CELL SURFACE ON VARIANT HAMSTER
GLYCOPROTEINS CELL LINES
H. CERI and J. A. WRIGHT Department
of Microbiology,
University (Receiced
of Manitoba,
15 Nocemher
Winnipeg.
Manitoba,
Canada
1978)
Resistance to concanavalin A was accompanied by modifications to the fundamental growth and membrane-associated properties of two concanavalin A-resistant cell lines which were independently selected by either single step or cycling procedures. 2. Using the galactose-oxidase [‘H]borohydride surface labelling procedure, several modifications in surface glycoproteins were detected on variant cells when compared to parental wild type cells; a prominent difference was the presence of an additional surface component, with an apparent molecular weight of N55,OOO on variant cells which was missing from both wild type populations. 3. These observations provide strong support for the view that modified cell surface glycoproteins are directly involved in the complex concanavalin A-resistant phenotype which includes changes to some fundamental biological properties; concanavalin A-resistant variants provide an opportunity to correlate changes in membrane function with modifications in membrane structure.
Abstract-l.
INTRODUCTION
plasma membrane of mammalian cells plays a key role in the regulation of a variety of cellular events. A great deal of evidence supports the view that the cellular membrane is intimately involved in such basic biological phenomena as cell growth, the immune response, intercellular communication, and cellular differentiation (Quinn, 1976; Nicholson, 1976). Also many biochemical differences at the cell surface have been found between normal and transformed cells; these differences appear to be important for expressing the potentially malignant properties of the transformed cell (Burridge, 1976). However much more information concerning relationships between membrane structure and function is required before a proper understanding of some of the complex regulatory mechanisms associated with the surface membrane is achieved. Recent advances in the field of somatic cell genetics have provided new approaches for investigating membrane properties (Till er al., 1973; Siminovitch, 1976). Correlations of functional changes with membrane structural alterations resulting from mutations can serve to clarify obvious, as well as subtle, details of structure-function relationships (Till er al., 1973). With this view in mind we isolated and characterized Chinese hamster ovary (CHO) cells which exhibit reslstance to the cytotoxic effects of the lectin, concanavalin A (Wright, 1973). This protein is being used as a tool to investigate many aspects of membrane biology (Brittiger & Schebli, 1976) since it interacts with specific oligosaccharide chains at the surface membrane (Sharon & Lis, 1972). Independently isolated concanavalin A-resistant cell lines generally exhibit a very complex phenotype which includes modifications in specific growth and membrane-associated properties. For example these lectin-resistant variants exhibit obvious temperature-sensitive growth properties, altered cellular morphology, increased sensitivity to some membrane-active agents, altered lectin aggluThe
545
tination properties, modified adhesiveness to substratum properties, altered lectin binding characteristics, and defective lectin-receptor mobility properties (e.g. Wright, 1973; Wright, 1975; Ceri & Wright, 1977a,b; Wright & Ceri, 1977; Ceri & Wright, 1978 a,b). Clearly, it is important to identify the surface modifications involved in concanavalin A resistance since it will lead to a better understanding of some fundamental principles of membrane function (Till et al., 1973). Recently, we used specific cell surface labelling techniques to examine the surface of a concanavalin A-resistant cell line in detail (Ceri & Wright, 1978a). The variant cells exhibited several changes in surface labelling patterns when compared to parenta! wild type cells: a major difference was the presence of an additional surface component, with an apparent molecular weight of 155,000 on resistant cells which was missing from wild type cells. When the galactose-oxidase-(‘H)borohydride technique was used the extra component accounted for 12-18”/, of the total labelled cell surface glycoprotein on resistant cells. Lactoperoxidase catalyzed iodination of surface polypeptides and the metabolic incorporation of labelled glucosamine into membranes of resistant and sensitive cells also revealed the presence of a high molecular weight surface structure on the concanavalin A-resistant but not sensitive cells. Therefore it seems that concanavalin A resistance is accompanied by alterations in cell surface glycoproteins, and that these changes are directly involved in determining the complex biological changes previously described for concanavalin A resistance (Ceri &Wright, 1978a). However, since much of the data presented to support this contention comes from a detailed study of the membrane glycoprotein content of a single concanavalin A resistant cell line, it is necessary to carry out further studies on the relationship between the complex concanavalin A resistant phenotype and cell surface glycoproteins with other independently selected resistant
H. CI KI and J. A. WRIGHT
546
and sensitive cell lines. As previously Wright. lated
197&t) variants
further are
very
establish
this interesting
describe
the results
studies
with
important correlation;
of such
a study
noted
(Ceri
independently
in order
to firmly
in this report and
& iso-
provide
we evi-
which strongly supports the idea that there is a direct relationship between the fundamental biological changes and an obvious alteration in surface
dence
glycoprotein
content.
\IATERI.ZI.S
A[\iD
METHODS
Chinese hamster ovary (CHO) cells were grown as suspension or monolayer cultures in cc-minimal essential medium at 34 (Flow Laboratories. Inc) supplemented with antibiotics and lo”,, (rol vol) fetal bovine serum (GIBCO Ltd) as described previously (Wright. 1973: Cerl & Wright. IY77a.b). Plating efiiciencies were determined by standard techniclues (Wright. lY73).
A concana\alin A-resIstant cell line was selected from a CHO population 1%hich was previously mutagenized with ethyl methane zulfonate (Eastman Kodak). Ceils (I x IO”) growing exponentlall) on ii IS x 100 mm culture plate at 34 were Ireatcd with 350 11 ml of the mutagen in growth medium ior 15 hr. The treated cells were washed with phosphate-bulfrrcd haline. resuspended in fresh growth medium without mutagen at 34 for 2 weeks 10 allow sur\ icing cell< to grow.. These cells were then subjected to ;I \ingle step-selectIon procedure by adding 1 x lOh mutaeenized cells to a I6 oz Brockway bottle (Brockway Glass ?o.) contalnlng 30 ml growth medium supplemented with 4Ojrg ml concana\alin A (Calbiochem. Co.). Note: since concanavalin A interacts with serum components in the 1973) the lectin-containing growth medium (Wright. medium has routinely Incubated at 34 for 24 h and the resulting precipitate was removed either by centrifugation or (iltrntlon: medium containing concanabalin A was always cleared of precipitate before being used In the selection of variant cells. After approximately t*o weeks in the presence of lrct~n the sur\i+ing cells were removed and the culture was cloned as described previously (Wright. 1~73). One clone was selected for further study and was referred to as \arlan-I (V-l): these cells here routine11 cultured in medium lacking concanavalin A 4 second ~arlant cell line wa:, selected from a non-mutageniLed wild type CHO population b! a cycling procedure (Wrlphr. lY73: Wright. lY75). Approximately I x IOh cells were added lo a lhor Brockua) bottle containing 30ml growth medium with 40~1.g ml concanavalin A and incubated al 34 for about 5 days. After treatment with lectin I Iable cell\ \\ere allowed 10 recover in the presence of normal growth medium lacking concana\alin A. After 7 IO days about I x IO” cells were again placed m a 160~ BI-ockwaq bottle contaming medium supplemented with 40 ,I@ ml concann\aI~n A. This procedure of exposing cells lo lectin followed by a recovery period was performed four times. Then from this point on cells were continuously exposed lo medium with concanavalin A: every time a monolayer formed the cells were removed and I x IOh cells were added to medium containins 40/l@ ml lectin. The population was exposed to concanavalin A-containing medium a total of I4 times. and was then cloned as prepiously described (Wright. 1973). A clone was selected and referred lo as \arlant-2 (V-2): these cells were cultured in medium lacking concanavalin A. After approximately 6 months of continuous culture in the absence of a selectee agent the two variant cell lines nere used 111the esperlmentc described in this report.
Cell surface labelling of galactose and galactosamlnc residues on cells previously grown in logarithmic suspcnsion culture was performed by the galactoxe oxidase-[2H]borohydride method (Cerl & Wright. 1978a; Cahmhcrg & Hakomori. 1973). Protein extraction and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoreais M;I\ carried out by the method of Laemmli (Laemmli. lY70). Gels were sliced into approximately I.5 mm scct~on\. digested in H,OZ, and counted in a toluene-based cocktarl with a scmtillation counter. Molecular weights were determined by comparing the distance a particular peak mo\ed in a gel with the distance that known molecular wcelght markers migrated In the gel (Cerl & Wright, 1977b: (‘en & Wright, 1978a). The markers that were used included serum albumin (69.000). ovalbumin (44.500). myoglohm (17.800). and cytochrome (’ (12.400). Also included \\cre the E. w/i RNA polymerase B’. B. n and r subunlts (gencrously provided by Dr C T. Chow) with molecular wclghtq of 165.000. 155.000. 95.000 and 39.000 respect iveIl.
Most biochemicals were purchased from the Sigma Chemical Co. and radio chemicals were obtained from New England Nuclear
RESULTS
The eRects of various concentrations of concana\aefficiency of the wild type and the lectin-resistant cell lines. V-l and V-2, were determined by incubating various numbers of cells for IO days at 34 in the presence of different concentrations of the lectin. and counting the number of colonies formed (Wright, 1973: Ceri & Wright. 1978b). It is obvious from Figs 1 and 2 that the concanavalin A-resistant lines are significantly more successful than the parental wild type populations at forming colonies in the presence of the lectin. For example. in the presence of 18pg! ml concanavalin A the wild tj pe populations showed a relative plating etficiency of lin A on the plating
IO 20
30 C 0
40 N
50 A
102030405 &q/ml
Fie. I. EtTec1 of Lariouc concentrations of concana\ 31111,\ on the plating efficiency of mild tlpc cell line\ in !hc absence and presence of the hapten. IO ’ M mcth>l Y-I)glucose. Wild type line from which V-l was selected. In the absence (0) and presence of hapten (0). Wild type line from which V-l was selected. in the absence (A) and pre\ence of hapten IA). Fig. 2. Effect of Larious concentrations of concana\.1/1n A on the plating efficiency of lectin-resistant cell line\ in rhe absence and presence of the hapten. IO-’ M methyl r-o-glucose. V-l in the absence (A) and presence (Cl ~lf hapten. V-2 in the absence (0) and presence I@) of hapt
