Cell Differentiation, 11 (1982) 135--140 135 Elsevier/North-Holland Scientific Publishers, Ltd.

CHARACTERIZATION OF THE F9 ANTIGEN(S) ISOLATED FROM TERATOCARCINOMA CELL CULTURE MEDIUM

P.J. McCORMICK, A. DIMEO, E. NEUNER and K. ARTZT

Laboratory of Developmental Genetics, Sloan-Kettering Institute for Cancer Research, New York, NY 10021, U.S.A.

(Accepted 28 December 1981)

Anti-F9 is a syngeneic antiserum directed against mouse teratocarcinoma cells which also reacts, by comple- ment-mediated cytotoxicity, with early mouse embryos and male germ cells. A molecule (or molecules) which specifically inhibits anti-F9, cytotoxicity can be recovered from the culture medium of undifferentiated terato- carcinoma cells. The inhibitory component is not present in the culture medium of differentiated teratocarcino- ma cells or embryonic fibroblasts. The inhibitory material binds to Ricinus communis I affinity columns indi- cating that it contains terminal non-reducing ~-galactosyl residues. The antigenicity of the molecule does not require protein, since the inhibitory activity is completely protease-resistant. Gel filtration indicates that the pro- tease digested inhibitory material has a molecular weight of more than 80,000.

F9antigen teratocarcinoma cell carbohydrates conditioned medium

1. Introduction

The 'F9-antigen', as originally defined (Artzt et al., 1973), is the target of the cyto- toxic antibody in a syngeneic serum raised against an undifferentiated teratocarcinoma cell line (F9). This antigen is detectable on all undifferentiated teratocarcinoma cells (in- cluding human (Holden et al., 1977)), early mouse embryos and male germ cells, but no t on any other cell type, including differenti- ated teratocarcinoma cells (Artzt et al., 1973). In fact, F9 cells that have been forced to differentiate by t rea tment with retinoic acid no longer react with anti-F9 sera (Strick- land and Mahdavi, 1978).

Despite repeated at tempts by a number of laboratories, the F9 antigen has never been isolated, and there has been some controversy regarding its biochemical composit ion (Vitet- ta et al., 1975; Muramatsu et ai., 1979; Buc- Caron and Dupouey, 1980). These studies utilized either immunoprecipi tat ion of whole- cell detergent lysates or enzyme digests of sur-

face molecules. In this report, we describe the isolation of the F9 antigen in a non<lenatured, intact form from the culture medium of un- differentiated teratocarcinoma ce l l lines. Since anti-F9 serum contains binding IgG antibodies (Damonneville et al., 1979) as well as the cytotoxic IgM antibody, we have fol- lowed purification of the F9 antigen(s) by inhibition of the cytotoxic activity of the orig- inal F9 serum. The F9 antigenic determinant is pronase-resistant and has a molecular weight greater than 80,000.

2. Materials and methods

2.1. Preparation o f conditioned medium

All cells were maintained as monolayer cul- tures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% glutamine, 1% penicillin and streptomycin. F9 (Artzt et al., 1973) and Nulli-SCC-1 (Martin and Evans, 1975) undifferentiated teratocarcinoma cells

0045-6039/82/0000---0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers, Ltd.

136

were maintained in 15% fetal calf serum (FCS); C3H primary cultures, obtained from fresh explants of 10--12 day old mouse em- bryos, were in 10% fetal calf serum (FCS), and PYS-2 cells from a parietal yolk sac car- cinoma (Lehman et al., 1974) were in 5% FCS. Confluent cultures were washed four times to remove absorbed serum components and then incubated in serum-free DMEM. After 16 h the medium was decanted, centri- fuged to remove cells and cellular debris, and concentrated by precipitation after being brought to 100% saturation of (NH4)2SO4. Radiolabeled conditioned medium (CM) was obtained via metabolic incorporation of radioactive precursors. In general, cells were incubated in full medium containing 1--3 t~Ci/ml of radioactive sugar and/or amino acid label for 24 h. They were then incubated for 16h in serum-free medium supplemented with the same amount of radioactive label. Unincorporated label was removed by dialysis. All radiochemicals were obtained from New England Nuclear ([14C]amino acid mixture, 55 mCi/m atom carbon; [z4C]galactose, 56.5 mCi/mmol; [3H]galactose, 14.2 Ci/mmol; [3H]glucosamine, 39.6 Ci/mmol), except [3SS]methionine (>500 Ci/mmol), which was purchased from Amersham.

2.2. Antisera and cytotoxicity assay

The anti-F9 sera were prepared according to published procedures (Artzt et al., 1973). All sera were tested by direct cytotoxicity; only those sera positive on F9 cells and nega- tive on PYS-2 and lymphocytes were used. Cytotoxic kill was determined by the trypan blue exclusion assay using rabbit complement as described in Artzt et al. (1973). To deter- mine inhibitory activity, a 40 ttl aliquot of CM was incubated with 20 t~l of anti-F9 for 1 h at 4°C. F9 cells and complement were then added and the entire mixture was rein- cubated at 37°C for 45 min before the addi- tion of trypan blue. Control experiments using anti-H2 with lymphocytes as the target cells established that the media conditioned

by F9 cells (F9-CM) inhibit the activity of the antiserum and not the complement (data not shown).

2.3. Aff inity chromatography

Ricinus communis aggiutinin-120 (RCA-I), which specifically binds terminal ~-D-galactose residues (Nicolson et al., 1974), was coupled to agarose according to the procedure of Lotan et al. (1974). The packed column was washed with 50 column volumes of the chro- matography buffer (0.01 M Tris-HC1, pH 7.2, 0.15 M NaC1). Aliquots of CM (0.5 ml) were allowed to bind to the column overnight at 4°C. The column was then washed with about 20 volumes of the Tris buffer and the specifi- cally bound material was eluted with this buffer containing 0.2 M D-galactose (Sigma).

2.4. Pronase digestion

A pronase (Calbiochem) solution was made fresh before each digestion at 100 mg/ml in the digestion buffer (50 mM Tris-HC1, pH 8.0, 10 mM CaC12) and incubated at 50°C for 1 h to inactivate contaminating glycosidases. The sample to be digested was in either phosphate- buffered saline (PBS) at pH 8.0 or the diges- tion buffer. 100 t~l of sample was incubated at 50°C with 10 t~l of the stock pronase solu- tion. Additional 10 ~1 aliquots of pronase were added at 24 h intervals for the next 120 h. At the end of this period, the reaction was stopped by boiling the samples for 1 h. Samples were then dialyzed against PBS; the material remaining had less than 1% of the original [3SS]Met or [3H]amino acid meta- bolic label.

2.5. Gel filtration

Samples were dissolved in 0.I M Tris-HCl (pH 8.0) plus 0.5 M NaCI and run on a 1.0 × 57.0 cm Sephacryl S-200 (Pharmacia) sizing column at a flow rate of 3 mlfn. The column had been calibrated with a variety of protein (cytochrome c, 12.5 K; ribonuclease A,

13.7 K; ovalbumin, 43 K; BSA, 68 K; and aldolase, 158K) and dextran (2000K, 79.4 K and 17.5 K) standards.

3. Results

Serum-free culture medium was coUected from confluent cultures of undifferentiated teratocarcinoma cells. The viability of the cells in serum-free medium for 16 h (94%) was not significantly different from the viabil- ity in serum-supplemented medium (96%) as measured by trypan blue dye exclusion. As shown in Fig. 1, the conditioned medium, after concentration via (NH4)2SO4 precipita- tion, completely inhibited the cytotoxic activ- ity of conventional anti-F9 serum. Condi- tioned medium collected from Nulli-SCC 1 cell cultures (Nulli-CM) had as much cytotoxic inhibitory activity per pg of protein (as deter- mined by absorbance at 280 nm or by the method of Lowry et al. (1951)) as that from F9 cell cultures (F9-CM). In contrast, at the same (see Fig. 1) or higher protein concentra- tion (data not shown), media conditioned by differentiated teratocarcinoma cells (PYS 2) or embryonic fibroblasts (C3H) had no inhibitory activity. Furthermore, differen-

I.O x to

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_ 0.6 X

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400 800 1600 3200 RECIPROCAL SERUM DILUTION

Fig. 1. Comparison of anti-F9 cytotoxic inhibitory activity of conditioned media from various cell lines. Conditioned media were adjusted to the same protein concentration (as determined by absorbance at 280 nm) prior to testing. Cytotoxic index (CI) is defined as (% dead in e x p e r i m e n t a l - % dead in complement control) + (100%--% dead in complement control). Positive control, no CM (e); C3H-CM (A); PYS-CM (~); F9-CM (o); Nulli-CM (X).

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TABLE I

Comparison of the anti-F9 cytotoxic inhibitory activity of Fg-CM from untreated and retinoic acid- treated F9 cell cultures

Cytotoxic index a

1/1600 b 1/3200

Experiment 1 F9-CM 0.23 0.06 RA-F9-CM c 0.54 0.51 Control d 0.65 0.52

Experiment 2 F9-CM 0.06 0 RA-F9-CM 0.51 0.07 control 0.50 0.27

a For definition of cytotoxic index, see legend to Fig. 1. b Dilution of anti-F9 serum. c Cells were treated with 10 -7 M retinoic acid (RA) for 4 days by which time 90% had differentiated as determined morphologically. Cultures were washed and incubated in serum-free medium which also con- tained 10- 7 M RA. After 16 h this medium (RA-Fg- CM) was collected and prepared as described in Ma- terials and Methods. The inhibitory activity of this material was compared to that of CM collected from duplicate cultures to which no RA had been added. d The control (no CM) represents maximum kill in each experiment.

tiation of F9 cells in the presence of retinoic acid resulted not only in the loss of the direct cytotoxic effect of anti-F9 sera against these cells (Strickland and Mahdavi, 1978), but also, as shown in Table I, in the loss of the anti-F9 inhibitory component in the culture medium. The small amount of residual activ- ity which remained was probably due to a subset of the population which does not dif- ferentiate under these conditions (Solter et al., 1979).

Since it had previously been reported that peanut agglutinin (PNA) could distinguish between F9 antigen-positive and -negative cells (Reisner et al., 1977), we attempted to further purify the antigen by the use of galac- tose-binding lectin affinity columns. The elu- tion profile of F9-CM or Nulli-CM run on Ricinus communis agglutinin I (RCA-I)

138

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O" I

I0

o~

o 'o

o

i ~ [ i I 20 50 40 50 60

FRACTION NUMBER

80

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Fig. 2. RCA-I co lumn elut ion profi le of F9-CM inhib- i tory activity. F9-CM, concen t ra ted via (NH4)2SO4 precipi ta t ion, was run on the ricin co lumn as de- scribed in Materials and Methods. Most of the pro- tein, as de te rmined by absorbance at 280 nm (_- e ) , was no t bound by the column. However , the ant i -F9 inhibi tory act ivi ty, as de te rmined by the percent live cells (o . . . . . -o) remaining in a cyto- tox ic assay, is bound by the co lumn and can only be e luted by the specific hapten , i.e. 0.2 M galactose. The arrow indicates the posi t ion at which con t inuous e lu t ion with 0.2 M galactose was commenced .

agarose columns, specific for terminal ~-D-

galactose residues (Nicolson et al., 1974), is shown in Fig. 2. The specifically bound material contained all of the anti-F9 inhib- itory activity. Neither the void volume nor

T A B L E II

Effec t of pronase digestion of ant i-F9 inhibi tory act ivi ty of F9 and NulIi-CM

cy to tox ic

Inhibi t ion index a

- -pronase +pronase b

F9-CM 1 c 0.94 0.76 2 0.84 0.89

NulIi-CM 1 0.73 0.90 2 0.89 0.90 3 1.00 0.89

a Inhibi t ion index (I.I.) is def ined as ( cy to tox ic index of posit ive con t ro l - - cy to tox i c index of exper imenta l ) - cy to tox ic index of posit ive control . b Al iquots of CM were split and subjected to similar t r ea tment excep t tha t pronase was added to one-half (as described in Materials and Methods) while only the buffer was added to the o ther half. c The numbers represent di f ferent lots of CM.

the wash material were inhibitory. This specif- ically bound material was greatly enriched for carbohydrate relative to protein. It contained approximately 18--20% of the input galactose label and 45--50% of glucosamine label but only 3--4% of the amino acid label ([3SS]Met or p4C]amino acid mixture).

T A B L E III

Sephacryl S-200 co lumn elut ion profi le of ant i -F9 cy to tox i c inhibi tory act ivi ty in F9-CM

Pool No. Cy to tox i c index a Inhibi t ion index b

A. Undigested CM 1 (19--22) c 0.04 d 0.93 2 (23--27) 0.46 0.18 3 (28--31) 0.48 0.14 4 (38--41) 0.56 0

B. Pronase-digested CM 1 (19--23) 0.06 d 0.90 2 (24--28) 0.50 0.20 3 (29--33) 0.66 0.03 4 (34--38) 0.62 0.05

a For def in i t ion o f Cy to tox i c index, see legend to Fig. 1. b Fo r def in i t ion o f Inhibi t ion index, see legend to Table II. c Numbers in parentheses indicate fract ions pooled. Each fract ion equals 1.0 ml. Void vo lume (Vo) was deter- mined by blue dext ran 2000 e lu t ion (Vo = 21 ml). d Ant i -F9 serum was used at a 1 /1600 di lut ion.

139

In fact, protein is no t required for F9 anti- genicity since the F9 antigen in condit ioned medium is completely resistant to extensive protease digestion (Table II). Incubation of F9 or Nulli-CM with pronase at 50°C for 5 days (see Materials and Methods) had no effect on its anti-F9 inhibitory activity. At the present time, however, we do not know whether there is any protein associated with the F9 antigen since the inhibitory activity of both undigested and pronase digested CM runs in the void volume of Sephacryl S-200 sizing columns (Table III). This would indicate a molecular weight, even for the pro- nase<ligested material, of greater than 80,000.

4. Discussion

We have partially purified the F9 antigen from the culture medium of F9 and NuUi- SCC 1 cell cultures. Further purification of the molecule was accomplished by passage over Ricinus communis agglutinin I affinity columns. The F9 antigenicity recovered from F9-CM, i.e. that material which contains the anti-F9 cytotoxic inhibitory activity, was completely resistant to protease digestion. At the present time, our evidence would indicate that the F9 antigen is a polysaccharide, gly- coprotein or proteoglycan with a molecular weight of at least 80,000.

Previous attempts to de f ine the biochemi- cal nature of the F9 antigen have led to con- flicting results. Originally, Vitetta and co- workers (1975) used whole anti-F9 serum to immunoprecipi tate radiolabeled antigens from detergent-solubilized F9 cell lysates. Sodium dodecyl sulfate-polyacrylamide gel analysis of this material indicated that the anti-F9 pre- cipitated two proteins with molecular weights of 44,000 and 22,000. Muramatsu et al. (1979), using similar procedures, confirmed this electrophoretic profile. Furthermore, they showed that the pronase-digested glyco- peptides from the immunoprecipitates were excluded from a G-50 sizing column, indicat-

ing a molecular weight greater than 6000. They concluded that these glycopeptides were released from the 44 and 22 kilodalton glyco- proteins. Our data do not support this con- clusion since the pronase<iigested inhibitory molecule(s) i'n F9-CM is excluded from an S-200 column (it would, of course, also be excluded from a G-50). This molecule must be greater than 80 kilodaltons and, therefore, could not have been derived from these smal- ler molecules as described by Muramatsu et al. (1979). Neither Vitetta nor Muramatsu a t tempted to inhibit the cytotoxic activity of anti-F9 with their immunoprecipitates; since F9 antisera contain binding IgG's as well as cytotoxic IgM antibodies (Damonneville et al., 1979), the 44 and 22 K proteins may be the targets of the non-cytotoxic IgG's.

Recently, Buc-Caron and Dupouey (1980) analyzed the cytotoxic inhibitory activity of material released from intact F9 cells by pa- pain digestion. They reported that limited papain digestion releases an anti-F9 inhibitory component that was excluded from a G-150 column. However, after more extensive diges- tion, the inhibitory material was included in the column and eluted at a position corre- sponding to a glycopeptide of 2--3 kilo daltons.

While it is difficult to reconcile their results with the data presented in this report, there are some possible explanations for the discrep- ancies. First, all of our digestion procedures were carried out at 50°C, since at this tem- perature contaminating glycosidases are inac- tivated whereas the pronase is not. Buc-Caron and Dupouey (1980) incubated their material with papain at 37°C and, therefore, the small glycopeptides they described may have been derived from the action of contaminating glycosidases. A second, more intriguing pos- sibility is that the F9 antigenic determinant may be present on more than one type of molecule; in their case, at tached to a surface- associated glycoprotein, whereas in ours, it may be a large polysaccharide or proteogly- can-type molecule secreted into the culture medium.

140

Acknowledgements

We thank Dr. Dorothea Bennett for help- ful criticism of the manuscript. This work was supported in part by grants from NIH.

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Note added in proof

Y. Iwakura has demonstrated that the F9 antigen isolated from whole F9 cell detergent lysates is also protease resistant and elutes in the void volume of an S-200 column (personal communication and manuscript submitted).