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Developmentally regulated surface structures of teratocarcinoma stem cells studied by mutant cell lines
Cell Differentiation, 15 (1984) 249-253
249
Elsevier Scientific Publishers Ireland, Ltd. CDF 00288
Developmentally regulated surface structures of teratocarcinoma stem cells studied by mutant cell lines P. Dr&ber a n d M. Vojtigkov/a Institute of Molecular Genetics. Czechoslovak Academy of Sciences, 142 20 Prague, Czechoslovakia
(Received 22 October 1984)
Monoclonal antibodies TEC-01, TEC-02, and TEC-03, which define three developmentally regulated antigens TEC-1 (SSEA-I-like), TEC-2, and TEC-3, have been used to isolate and characterize teratocarcinoma stem cell mutants with altered expression of surface glycoconjugates. Mutants lacking TEC-1 antigen have been isolated by exposing mutagenized P19SI801AI cells to TEC-01 antibody, which was conjugated to the toxin from Ricinus communis. None of the mutants exhibits significant changes in the expression of TEC-3 antigen, but some are defective in the expression of TEC-2 antigen. Analysis of the expression of TEC-I,2,3 antigens in different lectin-resistant F9 and OTF9-63 cell lines has shown that all express TEC-I antigen, but some lectin-resistant phenotypes exhibit reduction in the expression of TEC-2 and/or TEC-3 antigens. Mutational events in genes regulating the expression of specific glycosyltransferases or glycosidases appear to be the biochemical mechanism regulating the expression of TEC-1 and TEC-2 antigens. teratocarcinoma; mutant cells; lectins; monoclonal antibodies Introduction Developmentally regulated cell surface molecules have been viewed as candidates for regulators of cell-cell interactions and cell differentiation. Studies with plant lectins and monoclonal antibodies (MAbs) have identified a number of c a r b o h y d r a t e structures c o m m o n to murine teratocarcinoma stem cells and early embryonic tissues (Solter and Knowles, 1978; Muramatsu et al., 1979; Gooi et al., 1981). However, their biological significance has not been defined. One approach to elucidating the biosynthesis and developmental aspects of cell surface carbohydrates is to isolate and characterize mutant cell lines with altered expression of carbohydrate moieties of membrane glycoconjugates. We have successfully used two selection systems for single-
step isolation of such mutants. Cytotoxic plant lectins, from Triticum vulgaris (wheat germ agglutinin; WGA) and Ricinus c o m m u n i s (the toxin ricin; RIC), have been used to isolate a number of phenotypically different mutants derived from the F9 teratocarcinoma stem cell line (Dr~ber and Stanley, 1984b); here we report on the expression of three developmentally regulated antigens TEC1,2,3 (Dr~ber and Pokornh, 1984) in these mutants. Another set of mutants, derived from the parental cell line P19S1801A1 (McBurney et al., 1982) has been isolated by exposing mutagenized cells to TEC-01 antibody (Dr~ber and Pokorn/t, 1984) conjugated to ricin. These mutants exhibit significant changes in the expression of surface carbohydrates, as detected by altered antibody-binding properties and lectin-resistant phenotypes.
0045-6039/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland, Ltd.
250
Materials and Methods
Cell lines The origin, properties and culture conditions for F9 cells and cell lines derived from F9 have been described previously (Drfiber and Stanley, 1984a,b). P19S1801A1 cells (McBurney et al., 1982), PYS-2 cells (Lehman et al., 1974) and LEC12 cells (Chinese hamster ovary cell mutant expressing SSEA-1; Campbell and Stanley, 1983) were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and glucose (2.5 g/l).
Binding of monoclonal antibodies The IgM-class MAbs TEC-01, TEC-02 and TEC-03, which define TEC-1 (SSEA-l-like), TEC-2 and TEC-3 antigens, have been characterized previously (Dr/tber and Pokorn~, 1984). The antibodies were purified from ascitic fluid by ammonium sulfate precipitation and gel filtration. For direct RIAs, the antibodies were iodinated by the chloramine-T method and used as described elsewhere (Drfiber and Stanley, 1984a). In indirect RIAs the cells were incubated with MAbs (final concentration 1 /~g/ml) for 1 h at room temperature. Following three washes, the cells were incubated with 125I-labeled swine anti-mouse IgM (105 cpm/tube) for 1 h at room temperature, washed, collected, and counted in a gamma counter. Results show the averages of triplicate samples with cpm of negative controls subtracted (200-300 cpm).
TEC-O1-RIC conjugate In order to conjugate TEC-01 antibody and ricin, which was prepared by affinity chromatography and gel chromatography (Ho~ejgi and Kocourek, 1978), each of the proteins was reacted with a heterobifunctional crosslinking reagent, Nsuccinimidyl 3-(2-pyridyldithio)propionate (Pharmacia) according to Carlsson et al. (1978). After reduction, the TEC-01 SH-residues were reacted with the activated disulfide groups of ricin to form conjugates that were separated from free ricin by filtration on Sephacryl S-300 (Pharmacia) in phos-
phate-buffered saline (pH 7.4) containing 0.1 M lactose. SDS electrophoresis was performed according to Laemmli (1970) on 12% polyacrylamide slab gels (SDS-PAGE).
Selection of mutants Ethyl methanesulfonate (EMS)- or N-methylN-nitrosoguanidine (MNNG)-treated cells (Drfiber and Stanley, 1984b) were cultured for 6 days in a nonselective medium to allow expression of mutant phenotypes. The cells were harvested with trypsin-EDTA, washed and incubated for 2 h at room temperature in tissue culture medium containing TEC-01-RIC conjugate (5 /~g/ml) and lactose (0.1 M). After washing, the cells were seeded in culture medium plus lactose and cultured for 8 days. Control cultures were seeded to assess the plating efficiency of the cells.
Cytotoxicity of the conjugate and lectins The cells were either treated for 2 h with TEC0 1 - R I C conjugate and then cultured in medium containing 0.1 M lactose, or cultured for 8 days in the presence of lectins. The concentrations, which reduced relative plating efficiency to 10% (D10 values), were determined from the survival curves.
Results
Phenotypically different WGA- and RIC-resistant cell lines derived from F9 or OTF9-63 cells (Drfiber and Stanley, 1984b) were tested for the expression of TEC-1,2,3 antigens in a direct RIA (Table I). The iodinated antibodies exhibited binding properties identical to unlabeled ones: PYS-2 cells were labeled only by TEC-02 and TEC-03 antibodies, whereas LEC12 cells bound only TEC01 antibody. Representative clones of several lectin-resistant (Lec R) phenotypes were found to bind different amounts of these antibodies. The differences in the binding of TEC-01 antibody were less than two-fold, thus confirming our previous results (Dr/tber and Stanley, 1984b) that no significant changes in the expression of SSEA-1 accompany the Lec R phenotypic changes ex-
251
TABLE I Binding of TEC-01, TEC-02. lectin-resistant F9 cells Cell line
and TEC-03
% cpm in cell pellet
antibodies to
Lec R
TEC-0I
TEC-02
TEC-03
phenotype
OTF9-63 F9
42 40
24 40
31 30
Parent
OTF9.W2.7.9
34
21
OTF9.W3.12.5 OTF9.W3.12.6
27 30
6 8
13 14
W3
OTF9.R4.30.8 OTF9.R4.30.8.8
24 29
32 36
35 38
R4
F9.W5.1.11 F9.W5.1.5
39 40
33 37
18 12
W5
PYS-2 LEC12
0 42
41 0.3
1.5
W2
b
a 5 0.2
Controls
Cells (2.5x105) were incubated with ]25I-labeled antibodies (105 cpm) and the percentage of cpm bound was determined after separation of the cells from unbound antibody by centrifugation through a layer of 15% BSA.
pressed by W2, W3, R4, and W5 cells. The differences in the binding of TEC-02 and TEC-03 to some Lec R phenotypes were significant. When compared to parental cells, W3 cells bound less TEC-02 and TEC-03 antibodies and W2 cells bound less TEC-03 antibody. However, none of the mutants were deprived of TEC-2 or TEC-3 antigens completely. In an attempt to isolate mutants not expressing TEC-1 antigen we used another experimental approach in which TEC-01 antibody conjugated to ricin was employed as the selective agent. The conjugate was prepared as described above and separated from free ricin by gel filtration. Two elution peaks were obtained: the first contained T E C - 0 1 - R I C conjugate and the second free ricin (Fig. 1). Unreduced samples of T E C - 0 1 - R I C conjugate did not enter the separating gel (not shown), indicating that only covalently bound RIC was present. In the presence of lactose, which antagonizes the binding of the ricin B chain moiety to galactose residues on the cells (Oisnes and Pihl, 1982), the conjugate bound and exhibited cytotoxic activity to the cells expressing TEC-1 antigen
c
d
Fig. 1. S D S - P A G E of TEC-01-RIC conjugate and its components. The samples were: (a) TEC-01 antibody; (b) RIC; (c) the first peak from Sephacryl S-300 column; and (d) the second peak from Sephacryl S-300 column. The samples (10/Lg/line) were reduced with 2% mercaptoethanol.
(OTF9-63 and P19S1801A1) but not the cells negative for this antigen (PYS-2; Table II). When mutagenized P19S1801A1 cells were treated with TEC-01-RIC conjugate and then cultured in medium supplemented with lactose, surviving colonies were obtained at relatively high frequencies TABLE II Cytotoxicity and binding of TEC-01-RIC conjugate Cell line
Lactose added (M)
OTF9-63
0.1 -
PI9S180IAI
0.1 -
PYS-2
0.1
D]0 ~ (ng/ml) 20 1
25 3
> 10000 50
Binding to the cells (cpm × 10- 2) b 183 175 173 152 1 32
" The concentration of TEC-01-RIC conjugate, which reduced colony formation to 10% (D10 value), was calculated from individual surviving curves. b The binding to the cells (5×105 ) was determined in an indirect RIA; the concentration of TEC-01-RIC conjugate was 1 #g/ml.
252
TABLE III Selection of TEC-|- P19S1801A1 cells Mutagen (~ g/ml)
Frequency of surviving colonies
Cell lines isolated
for their ability to survive exposure to cytotoxic plant lectins. C o m p a r e d to wild-type cells, m u t a n t cells exhibited 2 - 5 - f o l d increased sensitivity to W G A and 3 - 7 - f o l d decreased sensitivity to RIC.
(X 10-6) a
EMS (100)
M N N G (2)
Discussion
<0.5 253
P19ST.I.1 P19ST.1.2 P19ST.1.3 P19ST.1.4 P19ST.1.5
56
Cells were treated with TEC-01-RIC conjugate as described in Materials and Methods. After 8 days, surviving colonies were counted. Survival frequencies were calculated from the number of colonies per number of cells plated corrected for plating efficiency in the absence of the conjugate. a
(Table III). N o surviving colonies were recovered a m o n g 2.2 × 10 6 unmutagenized cells challenged. Of the 15 colonies tested (see below), all were deficient in the expression of TEC-1 antigen. After recloning, five cell lines which retained a teratocarcinoma stem cell morphology have been established and tested for the expression of TEC-1,2,3 antigens (Table IV). All cell lines were negative for TEC-1 and positive for TEC-3 antigen. Some cell lines exhibited markedly reduced levels of TEC-2 antigen, thus suggesting heterogeneity a m o n g these mutants. The TEC-1 - phenotype was stable for at least 40 generations in the absence of selection. The mutant and parental cells were also compared TABLE IV Binding of TEC-01, TEC-02 and TEC-03 antibodies to P19S1801A1 mutants Cell line P19S1801A1 P19ST.l.1 P19ST.1.2 P19ST.1.3 P19ST.1.4 P19ST.1.5
cpm bound ( × 10 -2) a TEC-01
TEC-02
TEC-03
129 0 0 0 0 0
56 58 77 83 2 5
28 28 29 55 27 36
a The binding to the cells (5x10 5) was determined in an indirect RIA.
The experiments presented in this paper are part of a project aimed at elucidating the biosynthesis and developmental aspects of selected surface structures of embryonic cells by isolating and characterizing m e m b r a n e mutants of teratocarcinoma stem cells. Plant lectins and M A b s are mutually complementary tools for such analysis. Thus, the differences in expression of TEC-2 and TEC-3 antigens have been found a m o n g phenotypically distinct Lec R F9 cells, and T E C - 1 P19S1801A1 cell lines exhibited changes in the sensitivity to cytotoxic plant lectins. Direct coupling of a toxic lectin such as R I C to M A b directed against a developmentally regulated antigen produces a new toxic agent which allows selection of cells lacking that antigen. In fact, some M A b - R I C conjugates m a y be more useful than lectins for selecting mutants with altered expression of m e m b r a n e carbohydrates. Previously, we were unable to isolate RIC-resistant P19S1801A1 cells (Drfiber and Stanley, 1984b). However, when T E C - 0 1 - R I C conjugate was used, colonies composed of cells lacking TEC-1 antigen and resistant to R I C were recovered at relatively high frequencies (Table III). It has been hypothesized that in a differentiable system, such as teratocarcinoma stem cells, the expression of specific glycoconjugate structures at the cell surface m a y be intimately involved in differentiation p h e n o m e n a (Muramatsu et al., 1978, 1979). Thus the specific structures expressed at the cell surface reflect both the genetic capacity to produce such structures and the state of differentiation or determination of the cells. The increased frequency of T E C - 1 - cells following mutagenesis, the stability of the phenotype when the cells are grown under nonselective conditions and maintenance of their teratocarcinoma stem cell morphology suggest that the T E C - 1 - phenotype results from a mutational event rather than
253 f r o m s e l e c t i o n o f d i f f e r e n t i a t e d cells. R e c e n t l y , O T F 9 - 6 3 cells l a c k i n g S S E A - 1 h a v e b e e n i s o l a t e d b y e x p o s i n g m u t a g e n i z e d cells t o a n t i - S S E A - 1 a n t i b o d y a n d c o m p l e m e n t ( R o s e n s t r a u s et al., 1983). T h e s e cells a r e as s e n s i t i v e to t h e c y t o t o x i c l e c t i n s W G A a n d R I C as a r e t h e T E C - 1 mutants des c r i b e d here. T h i s s u g g e s t s t h a t s i m i l a r m u t a t i o n a l events, probably affecting expression or function o f c e l l u l a r g l y c o s y l t r a n s f e r a s e s o r g l y c o s i d a s e s , occ u r r e d in t h e s e cell lines. Q u a n t i t a t i v e e s t i m a t i o n b y t h e use o f W G A o f t h e f r e q u e n c y of r e v e r t a n t s to t h e c y t o t o x i c e f f e c t o f w h i c h t h e m u t a n t cells a r e m o r e s e n s i t i v e t h a n p a r e n t a l cells, a n d f u r t h e r g e n e t i c , b i o c h e m i c a l and biological analysis of these mutants should provide insight into the molecular mechanisms of such mutational events and into the biosynthesis and function of TEC-1 and TEC-2 antigens. Such a n a l y s i s m i g h t b e s i g n i f i c a n t l y a c c e l e r a t e d if t h e biochemical nature of the antigenic determinants recognized by monoclonal antibodies, which bind d i f f e r e n t l y to w i l d - t y p e a n d m u t a n t cell lines, is understood.
Acknowledgements W e t h a n k D r . P. S t a n l e y for k i n d l y p r o v i d i n g L E C 1 2 cells, Dr. F. F r a n ~ k f o r s w i n e a n t i - m o u s e I g M , a n d D r . V. Hoi'ejgi f o r ricin.
References Campbell, C. and P. Stanley: Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-I. Cell 35, 303 309 (1983). Carlsson, J., H. Drevin and R. Axen: Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723 737 (1978).
Dr~ber, P. and Z. Pokorn/t: Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies. Cell Differ. 15, 109 113 (1984). Drfiber, P. and P. Stanley: Cytotoxicity of plant lectins for mouse embryonal carcinoma cells. Somatic Cell Mol. Genet. 10, 435-443 (1984a). Dr/iber, P. and P. Stanley: Isolation and partial characterization of lectin-resistant F9 cells. Somatic Cell Mol. Genet. 10, 445-454 (1984b). Gooi, H.C., T. Feizi, A. Kapadia, B.B. Knowles, D. Solter and M.J. Evans: Stage-specific embryonic antigen involves al 3 fucosylated type 2 blood group chains. Nature (London) 292, 156 158 (1981). Ho~ejgi, V. and J. Kocourek: Studies on lectins. XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels. Biochim. Biophys. Acta 538, 299-315 (1978). Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 (1970). Lehman, J.M., W.C. Speers, D.E. Swartzendruber and G.B. Pierce: Neoplastic differentiation: characteristics of cell lines derived from a murine teratocarcinoma. J. Cell. Physiol. 84, 13-28 (1974). McBurney, M.W., E.M.V. Jones-Villeneuve, M.K.S. Edwards and P.J. Anderson: Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature (London) 299, 165-167 (1982). Muramatsu, T., G. Gachelin, M. Damonneville, C. Delarbre and F. Jacob: Cell surface carbohydrates of embryonal carcinoma cells: polysaccharidic side chains of F9 antigens and of receptors to two lectins, FBP and PNA. Cell 18, 183-191 (1979). Muramatsu, T., G. Gachelin, J.F. Nicolas, H. Condamine, H. Jakob and F. Jacob: Carbohydrate structure and cell differentiation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 75, 2315-2319 (1978). Olsnes, S. and A. Pihl: Toxic lectins and related proteins. In: The Molecular Actions of Toxins and Viruses, eds. P.L. Cohen and S. Van Heyningen (Elsevier/North-Holland Biomedical Press, Amsterdam) pp. 51-105 (1982). Rosenstraus, M.J.: Isolation and characterization of an embryonal carcinoma cell line lacking SSEA-1 antigen. Dev. Biol. 99, 318-323 (1983). Solter, D. and B.B. Knowles: Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-I). Proc. Natl. Acad. Sci. USA 75, 5565 5569 (1978).
249
Elsevier Scientific Publishers Ireland, Ltd. CDF 00288
Developmentally regulated surface structures of teratocarcinoma stem cells studied by mutant cell lines P. Dr&ber a n d M. Vojtigkov/a Institute of Molecular Genetics. Czechoslovak Academy of Sciences, 142 20 Prague, Czechoslovakia
(Received 22 October 1984)
Monoclonal antibodies TEC-01, TEC-02, and TEC-03, which define three developmentally regulated antigens TEC-1 (SSEA-I-like), TEC-2, and TEC-3, have been used to isolate and characterize teratocarcinoma stem cell mutants with altered expression of surface glycoconjugates. Mutants lacking TEC-1 antigen have been isolated by exposing mutagenized P19SI801AI cells to TEC-01 antibody, which was conjugated to the toxin from Ricinus communis. None of the mutants exhibits significant changes in the expression of TEC-3 antigen, but some are defective in the expression of TEC-2 antigen. Analysis of the expression of TEC-I,2,3 antigens in different lectin-resistant F9 and OTF9-63 cell lines has shown that all express TEC-I antigen, but some lectin-resistant phenotypes exhibit reduction in the expression of TEC-2 and/or TEC-3 antigens. Mutational events in genes regulating the expression of specific glycosyltransferases or glycosidases appear to be the biochemical mechanism regulating the expression of TEC-1 and TEC-2 antigens. teratocarcinoma; mutant cells; lectins; monoclonal antibodies Introduction Developmentally regulated cell surface molecules have been viewed as candidates for regulators of cell-cell interactions and cell differentiation. Studies with plant lectins and monoclonal antibodies (MAbs) have identified a number of c a r b o h y d r a t e structures c o m m o n to murine teratocarcinoma stem cells and early embryonic tissues (Solter and Knowles, 1978; Muramatsu et al., 1979; Gooi et al., 1981). However, their biological significance has not been defined. One approach to elucidating the biosynthesis and developmental aspects of cell surface carbohydrates is to isolate and characterize mutant cell lines with altered expression of carbohydrate moieties of membrane glycoconjugates. We have successfully used two selection systems for single-
step isolation of such mutants. Cytotoxic plant lectins, from Triticum vulgaris (wheat germ agglutinin; WGA) and Ricinus c o m m u n i s (the toxin ricin; RIC), have been used to isolate a number of phenotypically different mutants derived from the F9 teratocarcinoma stem cell line (Dr~ber and Stanley, 1984b); here we report on the expression of three developmentally regulated antigens TEC1,2,3 (Dr~ber and Pokornh, 1984) in these mutants. Another set of mutants, derived from the parental cell line P19S1801A1 (McBurney et al., 1982) has been isolated by exposing mutagenized cells to TEC-01 antibody (Dr~ber and Pokorn/t, 1984) conjugated to ricin. These mutants exhibit significant changes in the expression of surface carbohydrates, as detected by altered antibody-binding properties and lectin-resistant phenotypes.
0045-6039/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland, Ltd.
250
Materials and Methods
Cell lines The origin, properties and culture conditions for F9 cells and cell lines derived from F9 have been described previously (Drfiber and Stanley, 1984a,b). P19S1801A1 cells (McBurney et al., 1982), PYS-2 cells (Lehman et al., 1974) and LEC12 cells (Chinese hamster ovary cell mutant expressing SSEA-1; Campbell and Stanley, 1983) were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and glucose (2.5 g/l).
Binding of monoclonal antibodies The IgM-class MAbs TEC-01, TEC-02 and TEC-03, which define TEC-1 (SSEA-l-like), TEC-2 and TEC-3 antigens, have been characterized previously (Dr/tber and Pokorn~, 1984). The antibodies were purified from ascitic fluid by ammonium sulfate precipitation and gel filtration. For direct RIAs, the antibodies were iodinated by the chloramine-T method and used as described elsewhere (Drfiber and Stanley, 1984a). In indirect RIAs the cells were incubated with MAbs (final concentration 1 /~g/ml) for 1 h at room temperature. Following three washes, the cells were incubated with 125I-labeled swine anti-mouse IgM (105 cpm/tube) for 1 h at room temperature, washed, collected, and counted in a gamma counter. Results show the averages of triplicate samples with cpm of negative controls subtracted (200-300 cpm).
TEC-O1-RIC conjugate In order to conjugate TEC-01 antibody and ricin, which was prepared by affinity chromatography and gel chromatography (Ho~ejgi and Kocourek, 1978), each of the proteins was reacted with a heterobifunctional crosslinking reagent, Nsuccinimidyl 3-(2-pyridyldithio)propionate (Pharmacia) according to Carlsson et al. (1978). After reduction, the TEC-01 SH-residues were reacted with the activated disulfide groups of ricin to form conjugates that were separated from free ricin by filtration on Sephacryl S-300 (Pharmacia) in phos-
phate-buffered saline (pH 7.4) containing 0.1 M lactose. SDS electrophoresis was performed according to Laemmli (1970) on 12% polyacrylamide slab gels (SDS-PAGE).
Selection of mutants Ethyl methanesulfonate (EMS)- or N-methylN-nitrosoguanidine (MNNG)-treated cells (Drfiber and Stanley, 1984b) were cultured for 6 days in a nonselective medium to allow expression of mutant phenotypes. The cells were harvested with trypsin-EDTA, washed and incubated for 2 h at room temperature in tissue culture medium containing TEC-01-RIC conjugate (5 /~g/ml) and lactose (0.1 M). After washing, the cells were seeded in culture medium plus lactose and cultured for 8 days. Control cultures were seeded to assess the plating efficiency of the cells.
Cytotoxicity of the conjugate and lectins The cells were either treated for 2 h with TEC0 1 - R I C conjugate and then cultured in medium containing 0.1 M lactose, or cultured for 8 days in the presence of lectins. The concentrations, which reduced relative plating efficiency to 10% (D10 values), were determined from the survival curves.
Results
Phenotypically different WGA- and RIC-resistant cell lines derived from F9 or OTF9-63 cells (Drfiber and Stanley, 1984b) were tested for the expression of TEC-1,2,3 antigens in a direct RIA (Table I). The iodinated antibodies exhibited binding properties identical to unlabeled ones: PYS-2 cells were labeled only by TEC-02 and TEC-03 antibodies, whereas LEC12 cells bound only TEC01 antibody. Representative clones of several lectin-resistant (Lec R) phenotypes were found to bind different amounts of these antibodies. The differences in the binding of TEC-01 antibody were less than two-fold, thus confirming our previous results (Dr/tber and Stanley, 1984b) that no significant changes in the expression of SSEA-1 accompany the Lec R phenotypic changes ex-
251
TABLE I Binding of TEC-01, TEC-02. lectin-resistant F9 cells Cell line
and TEC-03
% cpm in cell pellet
antibodies to
Lec R
TEC-0I
TEC-02
TEC-03
phenotype
OTF9-63 F9
42 40
24 40
31 30
Parent
OTF9.W2.7.9
34
21
OTF9.W3.12.5 OTF9.W3.12.6
27 30
6 8
13 14
W3
OTF9.R4.30.8 OTF9.R4.30.8.8
24 29
32 36
35 38
R4
F9.W5.1.11 F9.W5.1.5
39 40
33 37
18 12
W5
PYS-2 LEC12
0 42
41 0.3
1.5
W2
b
a 5 0.2
Controls
Cells (2.5x105) were incubated with ]25I-labeled antibodies (105 cpm) and the percentage of cpm bound was determined after separation of the cells from unbound antibody by centrifugation through a layer of 15% BSA.
pressed by W2, W3, R4, and W5 cells. The differences in the binding of TEC-02 and TEC-03 to some Lec R phenotypes were significant. When compared to parental cells, W3 cells bound less TEC-02 and TEC-03 antibodies and W2 cells bound less TEC-03 antibody. However, none of the mutants were deprived of TEC-2 or TEC-3 antigens completely. In an attempt to isolate mutants not expressing TEC-1 antigen we used another experimental approach in which TEC-01 antibody conjugated to ricin was employed as the selective agent. The conjugate was prepared as described above and separated from free ricin by gel filtration. Two elution peaks were obtained: the first contained T E C - 0 1 - R I C conjugate and the second free ricin (Fig. 1). Unreduced samples of T E C - 0 1 - R I C conjugate did not enter the separating gel (not shown), indicating that only covalently bound RIC was present. In the presence of lactose, which antagonizes the binding of the ricin B chain moiety to galactose residues on the cells (Oisnes and Pihl, 1982), the conjugate bound and exhibited cytotoxic activity to the cells expressing TEC-1 antigen
c
d
Fig. 1. S D S - P A G E of TEC-01-RIC conjugate and its components. The samples were: (a) TEC-01 antibody; (b) RIC; (c) the first peak from Sephacryl S-300 column; and (d) the second peak from Sephacryl S-300 column. The samples (10/Lg/line) were reduced with 2% mercaptoethanol.
(OTF9-63 and P19S1801A1) but not the cells negative for this antigen (PYS-2; Table II). When mutagenized P19S1801A1 cells were treated with TEC-01-RIC conjugate and then cultured in medium supplemented with lactose, surviving colonies were obtained at relatively high frequencies TABLE II Cytotoxicity and binding of TEC-01-RIC conjugate Cell line
Lactose added (M)
OTF9-63
0.1 -
PI9S180IAI
0.1 -
PYS-2
0.1
D]0 ~ (ng/ml) 20 1
25 3
> 10000 50
Binding to the cells (cpm × 10- 2) b 183 175 173 152 1 32
" The concentration of TEC-01-RIC conjugate, which reduced colony formation to 10% (D10 value), was calculated from individual surviving curves. b The binding to the cells (5×105 ) was determined in an indirect RIA; the concentration of TEC-01-RIC conjugate was 1 #g/ml.
252
TABLE III Selection of TEC-|- P19S1801A1 cells Mutagen (~ g/ml)
Frequency of surviving colonies
Cell lines isolated
for their ability to survive exposure to cytotoxic plant lectins. C o m p a r e d to wild-type cells, m u t a n t cells exhibited 2 - 5 - f o l d increased sensitivity to W G A and 3 - 7 - f o l d decreased sensitivity to RIC.
(X 10-6) a
EMS (100)
M N N G (2)
Discussion
<0.5 253
P19ST.I.1 P19ST.1.2 P19ST.1.3 P19ST.1.4 P19ST.1.5
56
Cells were treated with TEC-01-RIC conjugate as described in Materials and Methods. After 8 days, surviving colonies were counted. Survival frequencies were calculated from the number of colonies per number of cells plated corrected for plating efficiency in the absence of the conjugate. a
(Table III). N o surviving colonies were recovered a m o n g 2.2 × 10 6 unmutagenized cells challenged. Of the 15 colonies tested (see below), all were deficient in the expression of TEC-1 antigen. After recloning, five cell lines which retained a teratocarcinoma stem cell morphology have been established and tested for the expression of TEC-1,2,3 antigens (Table IV). All cell lines were negative for TEC-1 and positive for TEC-3 antigen. Some cell lines exhibited markedly reduced levels of TEC-2 antigen, thus suggesting heterogeneity a m o n g these mutants. The TEC-1 - phenotype was stable for at least 40 generations in the absence of selection. The mutant and parental cells were also compared TABLE IV Binding of TEC-01, TEC-02 and TEC-03 antibodies to P19S1801A1 mutants Cell line P19S1801A1 P19ST.l.1 P19ST.1.2 P19ST.1.3 P19ST.1.4 P19ST.1.5
cpm bound ( × 10 -2) a TEC-01
TEC-02
TEC-03
129 0 0 0 0 0
56 58 77 83 2 5
28 28 29 55 27 36
a The binding to the cells (5x10 5) was determined in an indirect RIA.
The experiments presented in this paper are part of a project aimed at elucidating the biosynthesis and developmental aspects of selected surface structures of embryonic cells by isolating and characterizing m e m b r a n e mutants of teratocarcinoma stem cells. Plant lectins and M A b s are mutually complementary tools for such analysis. Thus, the differences in expression of TEC-2 and TEC-3 antigens have been found a m o n g phenotypically distinct Lec R F9 cells, and T E C - 1 P19S1801A1 cell lines exhibited changes in the sensitivity to cytotoxic plant lectins. Direct coupling of a toxic lectin such as R I C to M A b directed against a developmentally regulated antigen produces a new toxic agent which allows selection of cells lacking that antigen. In fact, some M A b - R I C conjugates m a y be more useful than lectins for selecting mutants with altered expression of m e m b r a n e carbohydrates. Previously, we were unable to isolate RIC-resistant P19S1801A1 cells (Drfiber and Stanley, 1984b). However, when T E C - 0 1 - R I C conjugate was used, colonies composed of cells lacking TEC-1 antigen and resistant to R I C were recovered at relatively high frequencies (Table III). It has been hypothesized that in a differentiable system, such as teratocarcinoma stem cells, the expression of specific glycoconjugate structures at the cell surface m a y be intimately involved in differentiation p h e n o m e n a (Muramatsu et al., 1978, 1979). Thus the specific structures expressed at the cell surface reflect both the genetic capacity to produce such structures and the state of differentiation or determination of the cells. The increased frequency of T E C - 1 - cells following mutagenesis, the stability of the phenotype when the cells are grown under nonselective conditions and maintenance of their teratocarcinoma stem cell morphology suggest that the T E C - 1 - phenotype results from a mutational event rather than
253 f r o m s e l e c t i o n o f d i f f e r e n t i a t e d cells. R e c e n t l y , O T F 9 - 6 3 cells l a c k i n g S S E A - 1 h a v e b e e n i s o l a t e d b y e x p o s i n g m u t a g e n i z e d cells t o a n t i - S S E A - 1 a n t i b o d y a n d c o m p l e m e n t ( R o s e n s t r a u s et al., 1983). T h e s e cells a r e as s e n s i t i v e to t h e c y t o t o x i c l e c t i n s W G A a n d R I C as a r e t h e T E C - 1 mutants des c r i b e d here. T h i s s u g g e s t s t h a t s i m i l a r m u t a t i o n a l events, probably affecting expression or function o f c e l l u l a r g l y c o s y l t r a n s f e r a s e s o r g l y c o s i d a s e s , occ u r r e d in t h e s e cell lines. Q u a n t i t a t i v e e s t i m a t i o n b y t h e use o f W G A o f t h e f r e q u e n c y of r e v e r t a n t s to t h e c y t o t o x i c e f f e c t o f w h i c h t h e m u t a n t cells a r e m o r e s e n s i t i v e t h a n p a r e n t a l cells, a n d f u r t h e r g e n e t i c , b i o c h e m i c a l and biological analysis of these mutants should provide insight into the molecular mechanisms of such mutational events and into the biosynthesis and function of TEC-1 and TEC-2 antigens. Such a n a l y s i s m i g h t b e s i g n i f i c a n t l y a c c e l e r a t e d if t h e biochemical nature of the antigenic determinants recognized by monoclonal antibodies, which bind d i f f e r e n t l y to w i l d - t y p e a n d m u t a n t cell lines, is understood.
Acknowledgements W e t h a n k D r . P. S t a n l e y for k i n d l y p r o v i d i n g L E C 1 2 cells, Dr. F. F r a n ~ k f o r s w i n e a n t i - m o u s e I g M , a n d D r . V. Hoi'ejgi f o r ricin.
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