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Cell surface binding affinity of Simian virus 40 T-antigen
VIROLOGY
117.173-185
(1982)
Cell Surface Binding Affinity of Simian Virus 40 T-Antigen JUTTA Department
of
LANGE-MUTSCHLER
Biochemistry,
AND
ROLAND
HENNING’
University of Ulm, P.O. Box .@66,0-7900 Ulm, Federal Republic of Germnnv Received July 23, 1981; accepted, October 20, 1981
Simian virus IO-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in wivo by the rejection of tumorigenic SV40transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SVQO-infected and -transformed cells express SV40 large tumor antigen (T-Ag) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 T-Ag solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of T-Ag to the cell surface (called “externally hound T-Ag”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive ‘%I-protein A assay using rabbit antisera directed against purified SV40 T-Ag. Serological analysis of T-Ag in cellular extracts and of externally bound T-Ag revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 T-Ag. We interpret our results as an indication that this property enables purified T-Ag to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.
INTRODUCTION
Simian virus 40 (SV40)-transformed cells express a SV40-specific tumor transplantation antigen (TSTA) encoded by the early region of SV40 DNA (for review see Tooze, 1980). TSTA is defined by cellular immune response demonstrated in vivo by the transplantation rejection test (Defendi, 1963; Khera et al., 1963; Habel and Eddy, 1963; Koch and Sabin, 1965) and in vitro by a T lymphocyte-mediated lysis of SV40-transformed cells (Tevethia et al., 1974; Tevethia and Tevethia, 1977; Trinchieri et al., 1976; Gooding, 1977; Knowles et al., 1979). The proteins known to be encoded by the early region of SV40 are large tumor antigen (T-Ag) which is located in the nucleus (Pope and Rowe, 1964) and small tumor antigen (t-antigen) which is found mainly in the cytoplasm (Prives et al., 1977). Several efforts to relate early SV40 pro’ Author dressed.
to whom
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173
teins to TSTA suggested that T-Ag or closely related antigens occur on the cell surface: (i) Using antisera directed against purified T-Ag, the presence of T-Ag or closely related antigens was demonstrated serologically on the surface of SV40-transformed cells (Deppert and Henning, 1979; Soule and Butel, 1979; Deppert et al., 1980; Soule et al., 1980; Henning et al., 1981; Lange-Mutschler et al., 1981). (ii) Biochemical analysis by cell surface ‘%I iodination using lactoperoxidase (Deppert and Henning, 1979) and of plasma membranes isolated from [35S]methionine-labeled SV40-transformed cells (Soule et al., 1980) demonstrated a T-Ag-like molecule with an identical electrophoretic migration behavior (MW = 94,000). (iii) Attempts using tsA mutants of SV40 failed to distinguish genetically between TSTA and nuclear T-Ag (Anderson et al., 1977), or between surface T and nuclear T-Ag (Deppert, 1980). (iv) The best evidence for the correlation between T-Ag and TSTA comes from the use of purified T-Ag which 0042~6822/82/030173-13$02.00/O Copyright 0 1922 by Academic Press, Inc. All rights of reproduction in any form reserved.
174
LANGE-MUTSCHLER
immunized mice against a SV40 tumor cell challenge (Chang et al., 19’77; Chang et al., 1979); in addition, immunization with purified D2, a T-Ag-related protein encoded by adeno 2-SV40 hybrid virus (Ad2+D2) protected mice against a challenge of syngeneic SV40 tumorigenic cells and induced in vivo the generation of cytotoxic T lymphocytes sensitized against SV$O-transformed cells as demonstrated in vitro by a cytotoxicity assay (Tevethia et al., 1980). It thus seems to be clear that large SV40 T-Ag possesses TSTA activity in regard to the induction of the cellular immune response in vivo and possibly to the formation of TSTA as a cell surface target antigen for cytotoxic T lymphocytes. At present, all these observations are compatible with the hypothesis that T-Ag is pleiotropic, i.e., multifunctional T-Ag seems to occur simultaneously in nuclei and on the cell surface. Yet, the obvious biological and biochemical questions concerning this unique property of T-Ag remained unanswered. The facts, however, that immunization with T-Ag solubilized and purified to homogeneity protected mice against the SV40 tumor challenge suggested a new and special property of T-Ag in terms of cell surface binding affinity. Therefore, we investigated the binding affinity of T-Ag to the cell surface of SV40-transformed and various other cell lines. In the present study, we provide evidence that T-Ag solubilized from SV40transformed or -infected cells binds in significant amounts to the surface of living monolayer cells. MATERIALS
AND
METHODS
Cell Lines and Vim~s Mouse Balb/c fibroblasts (3T3) and the corresponding SV40-transformed cells (SV3T3, SVTZ), the naturally transformed human epithelial cell line (HeLa) and the SV40-transformed human fibroblasts (SVSO), the hamster embryonal cell line (BHK) and the SV40-transformed hamster fibroblasts (H 65190 B, kindly provided by Dr. V. Defendi, New York Uni-
AND
HENNING
versity, New York), were grown in petri dishes or in Roux bottles in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 510% calf serum. African green monkey kidney cells (TC-7) were grown in DMEM supplemented with 5% fetal calf serum. Confluent TC-7 cells were infected with SV40 virus strain 777 (approximately 10 PFU/cell) for 24 or 48 hr. Sera (i) Rabbit anti-T-Ag serum. T-Ag extracted from SW0 cells and subsequently purified by immunoprecipitation and SDSpolyacrylamide gel electrophoresis was used for immunization of rabbits as described previously (Deppert and Henning, 1979). The titer against nuclear T-Ag of the rabbit anti-T-Ag serum used in the present study (No. 20) was determined by indirect immunofluorescence microscopy according to Pope and Rowe (1964) on ethanol-fixed SVT2 cells (T-Ag titer 1:3200). The titer against surface T was determined by the ‘251-protein A assay on living SVT2 cells (1:20). In order to abolish an unspecific binding activity of the rabbit anti-T-Ag serum No. 20 to the cell surface of living cells, this serum was absorbed once on ethanol-fixed 3T3 cells as described previously (Henning et al., 1981). The other sera were used without absorption. (ii) Hamster SVIO tumor serum. Hamster SV40 tumor sera were obtained from inbred C’Lak hamsters 3-6 weeks after subcutaneous injection with 2 X lo6 H 65/ 90 B cells as described previously (Henning et al., 1981). After screening for similarly high titers against nuclear T-Ag as determined by indirect immunofluorescence according to Pope and Rowe (1964) on ethanol-fixed SVT2 cells hamster SV40 tumor sera were pooled (hamster SV40 tumor serum pool 3, titer 1:lOOO). This pool had a low titer against surface T (1:lO) as determined by the ‘%I-protein A assay on living SVT2 cells. (iii) Rabbit anti-actin serum. Rabbit anti-actin serum was raised in rabbits immunized with purified actin of chicken
CELL
SURFACE
BINDING
OF SV40
T-ANTIGEN
175
Lange-Mutschler et al., 1981). Thirty micrograms of protein A (Pharmacia) was labeled for 10 min at room temperature with 1 mCi Nal? (NEN) in 0.5 ml PBS by the chloramine-T method according to Hunter and Greenwood (1962). ‘%I-Labeled protein A was purified by gel chromatography in PBS on Sephadex G-25. The specific radioactivities varied between 2 X 10’ and 4 X 10’ cpm/pg. Cells (2 X 103) harvested from monolayer cultures were seeded per well in Terasaki microtiter plates and kept overnight at 37” to become adherent. To study the cell surface binding activity of T-Ag 5 ~1 of cell extract corresponding to l-2 X lo5 cells was added to each well for 1 hr at 37”. In a separate set Cell Extracts of experiments we observed that the steady Confluent monolayer cells grown in Roux state of the uptake of T-Ag onto the surbottles or petri dishes were harvested me- face of living cells was achieved within 60 chanically by scraping the cells off using min. After removal of the extracts the a rubber policeman. After washing the plates were washed extensively by gentle cells three times with HEPES-buffered shaking in HEPES-DMEM over a period Dulbecco’s modified Eagle’s medium of 45 min including 12 changes of washing (HEPES-DMEM, pH 7.3) 2 X 10’ cells buffer. The wells were then incubated with (SVSO, HeLa, SV3T3, 3T3, H 65190 B, 5 ~1 of serum diluted 1:lO in HEPESBHK) or 4 X lo7 cells (SVTB) were sus- DMEM for 1 hr at 37”. After another sepended in 1 ml HEPES-DMEM. In order ries of washes the cells were incubated to inhibit the activity of proteases 100 ~1 with ‘%I-labeled protein A for 30 min at of Trasylol (Bayer) was added per one room temperature. Finally, the plates were milliliter of cell suspension. Cells were frowashed over a period of at least 2 hr with zen at -70” and kept in this form up to several changes of HEPES-DMEM until 3 months. Extracts of frozen cells were the background binding of lzjI-protein A prepared freshly for every experiment. to empty wells was reduced to approxiAfter thawing, the cell suspensions were mately 100 cpm. The wells were dried, cut homogenized at 4’ with a tightly fitting out of the plates, and counted in a gamma Dounce homogenizer. To assure that cells counter. Each set of Terasaki plates has were completely homogenized the suspento be examined for background binding sions were sonicated once at 4” for 30 set properties in the absence of cells in order and checked microscopically for the ab- to rule out an unspecific binding of extract sence of intact cells or nuclei. Cellular de- proteins, immunoglobulins, or ‘%I-protein bris and subcellular particles were re- A to the plastic surface. In the case of moved by ultracentrifugation at 105,000 unspecific binding it could be considerably g for 30 min at 4”. In the following the reduced (lo-20% of the binding to cells) supernatants are called “cell extracts.” by precoating the plates with 15% human serum albumin (HSA) solution overnight and by addition of 15% HSA to cell exlz51-Protein A Assay tracts, antisera, and ‘%I-protein A soluThe ‘251-protein A assay (Brown et al., tion. 1977) performed in the present study on As demonstrated by immunofluoresuntreated monolayer cells was modified as cence microscopy the cells remained viable described previously (Henning et al., 1981; and intact during the ‘?-protein A test.
breast muscles as described previously (Arnold et al., 1968; Henning et al., 1981). The titer against actin was determined by indirect immunofluorescence microscopy on ethanol-fixed 3T3 cells (titer 1:lOO). (iv) Rabbit anti-cell serum. Rabbit anticell serum was obtained from rabbits immunized intraperitoneally three times at 3-week intervals with 10’ H 65/90 B cells. This xenoantiserum (No. 15) has high titers as determined by indirect immunofluorescence microscopy on living ceils against cell surface antigens of various cell lines (H 65/90 B, SV80,3T3) (ranging between 1:3000 and 1:6000).
1’76
LANGE-MUTSCHLER
Using SV40-transformed cells and rabbit anti-T-Ag serum it could be shown that no more than 0.05% of the cells (i.e., one or two dead cells per well) became positive for nuclear T-Ag. Radiolabeled Cell Extracts Approximately 2 X lo6 SW30 cells grown in one petri dish (lo-cm diameter) were radiolabeled by incubation with 1 ml of methionine-free DMEM containing 40 &i of $S]methionine (Amersham) for 4 hr at 37”. After washing the cells were frozen and thawed once. Extracts of labeled cells (2 X 10’ cells/ml PBS) were prepared after homogenization at pH 7.3 as described above. The protein concentration in the cell extracts was determined by the method of Lowry (1951). In order to determine the specific radioactivity of the proteins, aliquots of the extracts were precipitated with ice-cold trichloroacetic acid (TCA, final concentration 10%) w/v), washed several times with ethanol at -2O”, dried, dissolved and counted in 10 ml of scintillation liquid (Quickszint, Zinsser, Frankfurt FRG). The specific radioactivity (cpm/ pg protein) of total protein in a representative experiment using radiolabeled extracts of SW30 cells (7 mg protein/ml) was 5 X lo5 cpm/mg. Immunoprecipitation Electrophoreti
and SDS-Gel
To check the T-Ag specificity for cell surface binding experiments 300 ~1 of extracts was incubated with 10 ~1 of hamster SV40 tumor serum, or with 10 ~1 of rabbit anti-T-Ag serum or with the corresponding normal sera. After incubation for 1 hr at 4” 100 ~1 of pelleted protein A-Sepharose (Pharmacia) preswollen in phosphatebuffered saline (PBS, pH 7.3) was added. After shaking gently for 1 hr at 4” the immunoprecipitates were pelleted by centrifugation. The supernatants were used to determine the cell surface binding affinity of T-Ag by the 12SI-protein A assay on SV3T3 and 3T3 cells. The immunoprecipitates were analyzed for T-Ag by SDSpolyacrylamide gel electrophoresis (SDSPAGE). After extensive washing with
AND
HENNING
NET buffer (0.15 M NaCl, 5 mM EDTA, 50 mM Tris-HCl, 0.5% NP40, pH 7.4) and finally with 50 mM NH4HC03, the immune complexes were eluted from the protein ASepharose using 500 ~1 of elution buffer (50 mM NH4HC03, 2% SDS, 5% 2-mercaptoethanol). The eluates were lyophilized, dissolved in 20 ~1 sample buffer (0.1 M Tris-HCl, pH 6.8, 5% 2-mercaptoethanol, 0.005% bromophenol blue) and then run on 12% SDS-polyacrylamide slab gels at a constant current of 12 mA (Laemmli, 1970). Proteins were visualized by staining with Coomassie blue. For the determination of the content of radiolabeled T-Ag [?S]methionine-labeled extracts of 2 X lo6 SV80 cells were immunoprecipitated with hamster SV40 tumor serum as described above. SDS-PAGE was performed on lo-cm 12% polyacrylamide gels in glass tubes (6-mm diameter) using a 1.5-cm stacking gel containing 5% acrylamide at a constant current of 3 mA/ tube at room temperature. The gels were frozen immediately at -2O”, cut into 2-mm slices, eluted overnight in 200 ~1 water, and counted for YS cpm in 10 ml scintillation liquid (Quickszint, Zinsser). Comparison of the ?S cpm of T-Ag and the specific radioactivity of the total cellular proteins (?S cpm/pg) after a 4-hr labeling period determined in the TCA precipitates revealed a T-Ag content of approximately 1.4 pg/ml equivalent to 0.1% of the total protein amount. This estimation was based on the average of specific radioactivity of total cellular proteins. RESULTS
Solubilixed SV40 T-Antigen Binds to the Cell Sqfkce of Various Cell Lines It was possible to demonstrate surface T on living SV40-transformed cells by immunofluorescence microscopy and by the ‘%I-protein A assay using sera from rabbits immunized with purified T-Ag and sera from SV40 tumor-bearing hamsters (Henning et al., 1981). As expected, in the present study incubation of three SV40transformed cell lines derived from three species (human SV80 cells, mouse SV3T3 cells, hamster H 65/90 B cells) with the
CELL
SURFACE
BINDING
rabbit anti-T-Ag serum resulted in a low but significant binding of ‘%I-protein A molecules (Table 1) confirming the presence of surface T on SV40-transformed monolayer cells in situ. The incubation of monolayer cells in situ with T-Ag-containing extracts from SV40-transformed cells led to a significant increase in the amount of anti-T-Ag antibodies and ‘*I-protein A bound to the surfaces of all cell lines tested by a factor ranging between 2 and 5 (P < 0.0005) (Table 1). In contrast, extracts from the naturally transformed human cell line (HeLa) did not alter the number of anti-T-Ag antibody binding sites on the surface of all three SV40-transformed cells tested (Table 1; P < 0.025). Thus, the number of surface T-like molecules on SV40-transformed cells was only increased by the external addition of surface binding T-Ag molecules (called externally bound T-Ag) from extracts of SV40-transformed cells, but not by an increase in the serological accessibility of original surface T. These observations were confirmed using extracts from 3T3 or BHK cells (data not shown). The T-Ag specificity of the anti-T-Ag serum binding activity is demonstrated by the subtraction of data obtained with normal rabbit serum from those obtained with rabbit anti-T-Ag serum. These results are proved statistically by Student’s t test. As outlined under Materials and Methods, after performing the ‘l-protein A assay the wells were routinely checked by immunofluorescence microscopy for the presence of nuclear TAg positive cells. Here, it should be pointed out that this examination of living monolayer cells for nuclear T-Ag was particularly important after incubation with cell extracts. After performing the extensive and careful washing procedure we could not detect more than 0.05% T-Ag-positive cells per well since dead cells disappeared during the washing steps. Additionally, the viability of target cells demonstrated by the impermeability for antibodies was assessed by using a rabbit antiactin serum under identical conditions, i.e., after incubation of living target cells with extracts (see Table 4). We can assume that the increase in the number of T-Ag bind-
OF SV40
T-ANTIGEN
177
ing sites corresponds to externally bound T-Ag. Thus, solubilized T-Ag in cell extracts has a cell surface binding affinity on SV40-transformed cells. The term affinity is used in a general sense to apply to binding between soluble T-Ag or T-Ag assembled with another component and binding sites on the cell surface. The presence of surface T on SV40transformed cells raised the question of possible binding between solubilized T-Ag and surface T as a putative receptor due to the occurrence of aggregates between native T-Ag molecules in solution (Carroll et al., 1974). Therefore, we extended these experiments using naturally transformed human (HeLa) and embryonic mouse and hamster cell lines (3T3, BHK) as target cells which do not expose T-Ag or serologically related surface antigens. As expected, all three cell lines were negative for surface T (Table 2). Again, incubation of these target cells with extracts from SV40-transformed cells of three different species (SVSO, SV3T3, H 65/90 B) led to a number of externally bound T-Ag molecules comparable with the number of surface T molecules on SV40-transformed cells as shown in Table 1. These data show that solubilized T-Ag does not need natural surface T present on SV40-transformed cells in order to bind to the cell surface. In conclusion, both sets of results shown in Tables 1 and 2 suggest a general cell surface binding affinity of soluble TAg or at least of certain T-Ag subclasses. A comparison of the data shown in Tables 1 and 2 suggests a stronger cell surface binding affinity of T-Ag to SV40transformed cells than to other cell lines. In order to test the possibility of a saturable interaction, we analyzed the cell surface binding affinity of T-Ag under identical conditions by varying the concentration of cellular extracts. Figure 1 shows that the increase of T-Ag-related molecules bound to the cell surface of SV3T3 cells reached an apparent saturation only at high concentrations, whereas on 3T3 cells comparable results were not yet obtained at these concentrations. Comparative analysis of the stability of externally bound T-Ag and surface T indicated
178
LANGE-MUTSCHLER
AND
TABLE
HENNING
1
'?-PROTEIN A ASSAY FOR THE BINDING AFFINITY 0~ T-Ag SOLUBUJZED FROM SV&TBANSFORMBD TOTHE CELLSURFACEOFSV~O-TRANSFORMEDMONOLAYERCELLS ‘“I-Protein
A molecules X lo-’ bound per microtiter
plate well
After incubation with cell extracts from different transformed cell lines Antisera Anti-T-Ag serum Normal rabbit serum
Target cells SVCQ
T-Ag specific binding Anti-T-Ag serum Normal rabbit serum
SV3T3
T-Ag specific binding Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
H 65/SO B
CELLS
SV40-
After incubation with DMEM
SVSO
sv3T3
984*34 532220
2571 f 337 640 + 108
24m1tm 434 2 101
1685 + 192 647 rt 113
1038 + 145 480+ 38
452 + 39
1931 + 353
1595 + 278
1038 * 222
558 + 149
327 k 24 141 + 23
1188 + 134 176 + 35
1252 * 174 314 i 42
1432 + 167 236+ 63
461 + 59 264k 62
186 + 33
1012 + 197
938 t 178
1136 r 178
193+
405 + 54 130 f 12
1274 r 310 ill? 17
780 f 103 139+ 28
880 r 122 149 t 21
n.d. n.d.
275655
1163 + 317
6415 106
731 + 123
n.d.
HG&OB
HeLa
85
Note. SVQO-transformed target cells (human SWO, mouse SV3T3, hamster H 65/3O B cells) were incubated for 1 hr at 37” with T-Ag-containing extracts prepared from SVIO-transformed cells (SVSO. SV3T3. H 65/9O B). Control experiments were performed witb an extract from HeLa cells or only with HEPES-buffered DMEM. After washing. the cells were incubated with 1:lO dilutions of rabbit anti-T-Ag- of normal rabbit serum in HEPES-buffered DMEM and finally with ‘“I-protein A as described under Materials and Methods. The results represent the ‘“I-protein A molecules X lo-’ (mean + SD, N = 10) per one Terasaki microtiter plate well seeded with 2 X lo’ cells. The T-Ag specific binding indicating surface T and externally bound T-Ag was calculated by the formula: IS - NS f (SD IS* + SD NSP)1/2, IS is the number of protein A molecules bound to cells incubated with rabbit anti-T-Ag serum; NS is the number of protein A molecules bound to cells incubated with normal rabbit SW”lll
that both types of antigens stayed stable up to 24 hr after precoating with anti-TAg sera (data not shown). Recently it was reported, that T-Ag expressed in SV40-transformed cells exists in various species having different biochemical properties when compared with T-Ag expressed in SV40-infected monkey kidney cells (Kress et al., 1979). We therefore tested whether T-Ag solubilized from SV40-infected TC-‘7 cells express a cell surface binding affinity comparable to that of T-Ag extracted from SV40-transformed cell lines. As shown in Table 3, T-Ag extracted from TC-‘7 cells SV40 infected for 24 or 48 hr showed a cell surface binding affinity to mouse 3T3 cells similar to T-Ag from SV40-transformed cells. In control experiments, extracts from noninfected TC-7 cells did not alter the binding of antiT-Ag antibodies to the cell surface of 3T3 cells. As expected from the increase of the production of T-Ag the amount of externally bound T-Ag obtained 48 hr p.i. increased significantly when compared with the binding data determined 24 hr p.i.
Similar results were obtained using TC-7 cells as target cells for extract incubation (data not shown). These data clearly demonstrate, that there was no distinct difference in the cell surface binding affinities between T-Ag extracted from SV40transformed and from lytically SV40-infected cells. Externally Bound and Soluble T-Ag Molecules Are Serologicall~ Similar
The data presented so far strongly suggest that the increase of surface T molecules on cells incubated with extracts from SV40-transformed or -infected cells is due rather to the binding of T-Ag or closely related SV40-specific molecules than to the binding of serologically cross-reacting but cellular proteins which are expressed merely in SV40-transformed cells. This interpretation was checked by examining whether cell extracts were still able to increase the number of surface T molecules after specific removal of T-Ag by immunoprecipitation. To extend the serological
CELL SURFACE
BINDING
179
OF SV40 T-ANTIGEN
TABLE 2 ‘=I-PROTEIN A ASSAY FOR THE BINDING AFFINITY OF T-Ag SOLUBILIZED FROM SV40-TRANSFORMED CELLS TO THE CELL SURFACE OF HeLa, 3T3 AND BHK CELLS ‘?-Protein
A molecules X 10m6bound per microtiter
well
After incubation with cell extracts from different SV40-transformed cell lines Antisera
Target cells
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
HeLa
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
3T3
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
BHK
After incubation with DMEM 352 1- 54 + 32 97 f 62
255
SV80
H 65/90 B
SV3T3
993 -t 234 f 56 667 f 240 326
736 f 112
938 rf: 164
422f
46
432k
314
Lk 121
506
67
?I 177
174 + 22 50 f 49
935 + 143 194 + 36 741 + 147
603 150
+ 81 f 18 453 f 83
361f 61+ 300
f
29
140 f 27 103 + 20 37 zk 33
620 f 150 * 470 *
644k 42 279 f 33 365 + 53
538 277
f f
22
224f44
87
30 92
261+
26 12
91 94
Note. Human HeLa, mouse 3T3, hamster BHK cells were incubated with T-Ag containing extracts prepared from different SV40-transformed cell lines (SVSO, SV3T3, H 65190 B) or with HEPES-buffered DMEM as control. T-Ag molecules externally bound to the cell surfaces were determined by incubation with rabbit anti-T-Ag serum or normal rabbit serum and ‘?-protein A. The results represent the number of ‘%I-protein A molecules X 10m6(mean + SD, N = 10) bound per one Terasaki microtiter plate well seeded with 2 X lo3 cells. The T-Ag specific binding was calculated in the same way as for Table 1.
specificity of this type of experiment we used two different types of anti-T-Ag sera: hamster SV40 tumor serum (HaT, pool No. 3) for immunoprecipitation of T-Ag from cell extracts and rabbit anti-T-Ag serum for the measurement of externally bound T-Ag. A representative experiment is shown in Fig. 2: Aliquots of an extract of SW30 cells were immunoprecipitated with either hamster SV40 tumor serum or normal hamster serum (NHS). The immunoprecipitates were analyzed by SDSPAGE, and, as expected, hamster SV40 tumor sera precipitated T-Ag while NHS did not recognize T-Ag (Fig. 2B). The corresponding supernatants of both immunoprecipitates were then applied for the cell surface binding assay on SV3T3 cells under standard conditions using rabbit anti-T-Ag serum. Figure 2A shows that the typical increase in the binding of antiT-Ag serum on SV3T3 cells after incubation with SW30 cell extract preprecipitated with normal hamster serum was not al-
tered when compared with SV3T3 cells incubated with the untreated extract. In contrast, incubation of SV3T3 cells with SW30 cell extract preprecipitated with hamster SV40 tumor serum did not lead to an increase in the T-Ag specific binding when compared with cells incubated merely with medium. Similar results were obtained using 3T3 as target cells for a TAg cell surface binding assay (data not shown). These results confirm the data presented in Tables l-3 and moreover, they allow two conclusions in regard to the specificity of the cell surface binding affinity of T-Ag molecules from cell extracts: (i) removal of T-Ag from cell extracts by immunoprecipitation was clearly correlated with the disappearance of the cell surface binding affinity of T-Ag; (ii) using hamster SV40 tumor serum for the immunoprecipitation of T-Ag and rabbit anti-T-Ag serum for the determination of externally bound T-Ag molecules indi-
180
LANGE-MUTSCHLER
without extract
extract
dilution
FIG. 1. Binding of T-Ag to 3T3 cells and SV40transformed monolayer cells (SV3T3) at different concentrations of SV3T3 cell extracts. Mouse fibroblasts (3T3) and the corresponding SV40-transformed cells (SV3T3) were incubated with different dilutions of an extract prepared from SV3T3 cells. The concentration of undiluted extract (2 X lo7 SV3T3 cells/ml) was the same as used for the experiments described in Tables l-3. T-Ag molecules present on the cell surfaces were detected using 1:lO dilutions of rabbit anti-T-Ag serum or normal rabbit serum and ‘251-protein A. The data represent the T-Ag specific binding calculated in the same way as for Table 1 (N = 6).
cated the serological cross-reaction of both types of antisera. This situation corresponds exactly to the previously described cross-reacting specificities of both anti-TAg antibodies used for the demonstration of surface T on SV40-transformed cells (Henning et al., 1981). The observation of the cell surface binding affinity of T-Ag in the presence of total cell extracts prompted the obvious question as to whether other cellular proteins might also bind in comparable amounts to the surface of monolayer cells in situ. Although, due to the reasons mentioned above, there seems to be no doubt about the cell surface binding affinity of T-Ag, other proteins might influence the binding affinity of T-Ag. Due to the extensive rinsing procedure (see Materials and Methods) designed to wash off cellular proteins and simultaneously to maintain monolayer target cells adherent, presumably several cellular proteins present in the cell ex-
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HENNING
tracts might bind to the surface of target cells. Therefore, we examined the binding of cellular proteins by three approaches. (i) The amount of proteins bound to the surface of target cells was estimated using radiolabeled extracts of cells. In a representative experiment we determined the specific radioactivity of total cellular proteins in [?S]methionine-labeled SW30 cell extracts (5 X lo5 ?S cpm/mg). The incubation of SV80 target cells with these extracts (1.8 X lo4 ?S cpm/well corresponding to 35 pg protein/well) did not lead to a measurable amount of $S]methioninelabeled protein bound to the target cells (20 f 17 cpm/well). This result indicated that no cellular protein bound to the cell surfaces was detectable under these experimental conditions. (ii) Binding properties of various proteins to the cell surface were estimated using polyspecific rabbit antisera directed against hamster cells (H 65/90 B). This antiserum has a high titer against cell surface antigens on several cell lines (see Materials and Methods). A considerable number of binding sites of these- polyspecific antibodies was detected on untreated SVT2 cells but no significant increase was observed after incubation of these cells with extracts of the same cells (P < 0.475) (Table 4). (iii) The cell surface binding affinity of actin as a good example of a major intracellular protein was determined by using a rabbit antiactin serum. The data presented in Table 4 show that in comparison with cells incubated with medium, no significant amounts of both types of antibodies were bound to the cell surface after incubation of target cells with cell extracts. In summary, despite the sensitivity of the assay and the specificity of the antisera used none of these data indicate a significant increase in ceil surface binding of cellular proteins after incubation of living target cells with extracts. DISCUSSION
Simian virus 40 T-Ag appears to bind in significant amounts to the cell surface of various transformed and nontransformed cell lines. This finding helps to explain the mechanism by which immuni-
CELL SURFACE BINDING
181
OF SV40 T-ANTIGEN
TABLE 3 ‘z-I-P~~~~~~
A ASSAY FOR THE BINDING AFFINITY OF T-Ag SOLUBILIZED FROMSV40-INFECTED TC-7 CELLS TOTHE CELLSURFACEOF~T~ CELLS ‘?-Protein
Antisera Anti-T-Ag serum Normal serum T-Ag specific binding
3T3 cells incubated with DMEM
A molecules X 10m6bound per microtiter
plate well
3T3 cells incubated with extract of TC7 cells
3T3 cells incubated with extract of SV40-infected TC-7 cells (24 hr p.i.)
3T3 cells incubated with extract of SV40-infected TC-7 cells (48 hr p.i.)
194 f 52 170 Ik 45 24 + 69
433 f 75 190 f 29 243 f 80
947 + 252 248 f 48 699 + 256
248+40 252 + 72 -4 + 82
Note. Mouse 3T3 cells were incubated with T-Ag-containing extracts prepared from TC-‘7 cells infected with SV40 virus for 24 or 48 hr. As a control, 3T3 cells were incubated with uninfected TC-7 cells or only with growth medium. T-Ag molecules externally bound to the cell surfaces were determined by incubation with rabbit anti-T-Ag serum or normal rabbit serum and ‘251-protein A. The results represent the number of ‘%I-protein A molecules X 10e6 (mean * .SD, N = 6) bound per one Terasaki microtiter plate well seeded with 2 X l@ cells. The T-Ag specific binding was calculated in the same way as for Table 1.
zation with purified T-Ag induces the rejection of SV40-induced tumors in mice. At the moment, we have no serological or biochemical data hinting at any significant differences between native surface T as detected on SV40-transformed cells in situ and externally bound T-Ag. Hence, the TSTA activity of highly purified T-Ag might be caused by its cell surface binding affinity. The evidence for cell surface binding affinity of T-Ag in extracts from SV40transformed cells is established by several qualitative and semiquantitative results: (i) T-Ag from SV40-transformed or -infected cells binds to different cell lines independent of the species and the cell transformation. (ii) Removal of T-Ag from cell extracts by immunoprecipitation led to an abolishment of its cell surface binding activity. Using two different types of anti-T-Ag sera demonstrates the serological similarity between T-Ag immunoprecipitated from all extracts and externally bound T-Ag. (iii) Extracts from non-SV40transformed cells do not alter the amount of surface T on SV40-transformed cells. Based on these observations we have to reconsider possible differences and simi-
larities between surface T and nuclear TAg as discussed previously by ourselves and other laboratories. As pointed out in the Introduction, there is so far no genetic or biochemical evidence for molecular differences between both forms of T-Ag. Analyzing the presence of surface T on formaldehyde-fixed cells by immunofluorescence microscopy using SV40 tumor sera or rabbit anti-T-Ag sera, we previously described serological differences in comparison with nuclear T-Ag in cells fixed with formaldehyde followed by methanol/ acetone treatment (Deppert et al., 1980). This interpretation was principally not compatible with observations made on living cells. Soule et al. (1980) described unique antigenic determinants of surface T on living cells, but only after putting monolayer cells into suspension with EDTA or after storing these cells in suspension in 90% buffered glycerol at -30”. However, using an ‘%I-IgG-blocking assay showed that antisera specifically directed against surface T as well as SV40 tumor sera blocked the cell surface binding of rabbit antisera directed against purified and denatured T-Ag (Soule et al., 1980). Similarly, sera against purified T-Ag could
LANGE-MUTSCHLER
AND HENNING
B extract
+
extract
extract Drecbitated with HaT
HaT
NHS
NHS
25
FIG. 2. Cell surface binding activity of T-Ag in cell extracts peprecipitated with anti-T-Ag serum. Aliquots of extract prepared from SV30 cells were immunoprecipitated with undiluted hamster SV40 tumor serum (HaT) or with normal hamster serum (NHS) and protein A-Sepharose. (A) iz51Protein A assay. Untreated extract and the supernatants of the immunoprecipitates were analyzed by the ‘=I-protein A assay for the presence of cell surface binding T-Ag molecules: SV3T3 cells were incubated with untreated extract (+ extract), extract immunoprecipitated with hamster tumor serum, extract immunoprecipitated with normal hamster serum, or incubated with growth medium as a control (- extract). Externally bound T-Ag was determined using rabbit anti-T-Ag serum or normal rabbit serum and ‘?-protein A. The data represent the T-Ag specific binding (mean f SD, N = 6) calculated in the same way as for Table 1. (B) SDS-polyacrylamide gel electrophoretic analysis of unlabeled immunoprecipitates. The immunoprecipitates were analyzed on a 10% SDSpolyacrylamide slab gel. The proteins were stained with Coomassie blue.
be demonstrated to block the binding of hamster SV40 tumor sera to cell surfaces (Henning et al., 1981). These observations indicated directly the very similar properties of different antisera with respect to the specificity for surface T. At present, there is no direct evidence available for serological dissimilarities between surface T and nuclear T-Ag. It should be noticed that T-Ag extracted and enriched from nuclei isolated from SV40-transformed cells possesses a potent TSTA activity (Rogers et al., 1977). Both observations, the cell surface binding affinity of solubilized T-Ag and that surface T is serologically indistinguishable from nuclear T-Ag raise the question whether surface T occurs as a membraneassociated or -integrated protein. For the latter possibility one has to assume the
classical signal sequence mechanism (Blobe1 and Sabatini, 1971; Blobel and Dobberstein, 1975) or the trigger hypothesis suggesting an internal sequence-mediated membrane insertion (for review see Wickner, 1980). The amino acid sequence of TAg derived from the nucleotide sequence of SV40 DNA (Fiers et al., 1978; Reddy et al., 1978) does not seem to provide sufficiently extensive hydrophobic regions complying with the trigger hypothesis nor with the signal sequence. Alternatively, Mark and Berg (1979) reported a new splicing region in early SV40 RNA that would be able to code for a T-Ag with a hydrophobic carboxy terminus (T*-Ag). However, according to careful tryptic peptide analyses of T-Ag extracted from SV40 wild-type and dl 1263 mutant-infected cells an apolar region at the carboxy ter-
CELL SURFACE
BINDING TABLE
4
‘%I-protein
Antiserum Rabbit anti-cell serum Rabbit antiactin serum Normal rabbit serum
183
OF SV40 T-ANTIGEN
A molecules X 1O-6 bound per microtiter plate well
After incubation with DMEM 108,319 + 8091 131 + 11 117 * 45
After incubation with extract of SVT2 cells 112,642 _+ 6942 110 f 21 75f 15
Note. SV40-transformed monolayer cells (SVT2) were incubated with an extract prepared from SVT2 cells or with HEPES-buffered DMEM. Cellular proteins present on the cell surface were measured using polyspecific rabbit anti-cell serum and ‘%I-protein A. Actin molecules from cell extracts externally bound to the cell surface were assayed using antiactin serum and lz51-protein A. The data represent the number of protein A molecules X 1O-6 (mean + SD, N = 6) bound per one Terasaki microtiter plate well seeded with 2 X lo3 cells.
minal end was not detectable (Denhardt and Crawford, 1980). For these and other reasons requiring a variety of distinct biochemical properties for integral membrane proteins it seems to be unlikely that T-Ag or subclasses (surface T) belong to the family of integrated membrane proteins. Although evidence against regarding surface T as an integrated membrane protein remains to be determined, at the moment, for several reasons we favor considering surface T as a membrane-associated protein. First, both the present observations about the cell surface binding affinity and the induction of the TSTA activity by solubilized T-Ag are compatible with the idea that surface T is a membraneassociated protein. Second, the serologitally detectable similarity between surface T, externally bound T-Ag, and nuclear T-Ag indicates the possibility that surface T arises by leaking of T-Ag from living or dead cells. This idea is supported by the detection of soluble T-Ag in the growth medium of internally radiolabeled cells (umpublished observations). The question as to whether this mechanism causes the pleiotropic expression of T-Ag in nuclei and on the cell surface remains to be answered. According to our present knowledge our
findings can only suggest that T-Ag has a certain cell surface binding affinity without allowing us to define the type of binding sites. Since the binding experiments were performed by using extracts obtained from SV40-transformed and -infected cells but not with enriched or even highly purified T-Ag we do not know whether T-Ag or certain subclasses bind directly to unspecific binding sites, to putative receptors, or alternatively, whether T-Ag binds indirectly by using ligands such as nonviral T-Ag or nucleic acids. It should be noticed that the relatively high concentrations of cell extracts are necessary with respect to the total capacity of cell extracts to demonstrate the presence of externally bound T-Ag (see Fig. 1). One explanation of this event might be an methodologically intrinsic aspect: the total amount of T-Ag available for cell surface binding events might be considerably reduced during the preparation of cellular extracts under physiological conditions (i.e., ionic strength, pH, absence of denaturing agents) by removing the cellular debris including the plasma membranes with externally bound T-Ag by centrifugation. In interpreting our findings in terms of their possible immunological consequences it is striking to recognize that the
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activity of highly purified and soluble TAg to stimulate a SV40-specific cellular immune response in mice can in fact be correlated with a cell surface binding property. It has recently been shown that incubation of spleen cells with soluble proteins such as trinitrophenylated bovine serum albumin or ovalbumin renders these cells susceptible to lysis by hapten-specific cytotoxic T lymphocytes (Schmitt-Verhulst et al., 1978; Ballas and Henney, 1979). It thus would be interesting to know whether the attachment of T-Ag, released from SV40-transformed cells, to normal cells might lead to the formation of a SV40 tumor-specific transplantation antigen. ACKNOWLEDGMENTS
The authors thank Ms. Cornelia Jenkner for technical assistance and Mrs. Madge B. Henning for the help with the manuscript. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (He 882/4 and He 882/5-3). REFERENCES ANDERSON, J. L., MARTIN, R. G., CHANG, Ch., MORA, P. T., and LIVINGSTON, D. M. (1977) Nuclear preparation of SV40 transformed cells contain tumor specific transplantation activity. Vidogg 76, 420425. ARNOLD, H., HENNING, R., and PETTE, D. (1968). Binding of glycolytic enzymes to structure proteins of the muscle. Eur. J. Biochem 6, 163-171. BALLAS, Z. K., and HENNEY, C. S. (1979). Generation of H-2 restricted cytotoxic T cells by trinitrophenylated proteins in vitro: Specificity and requirements. .I ImmunoL 123,1696-1704. BLOBEL, G., and SABATINI, D. D. (1971). Ribosomemembrane interaction in eucaryotic cells. Bimnembrunes 2,193-195. BLOBEL, G., and DOBBERSTEIN, B. (19’75). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. CelL BioL 67,852862. BROWN, J. P., KLITZMAN, J. M., and HELLSTR~~M, K. E. (1977). A microassay for antibody binding to tumor cell surface antigens using ‘%I-labelled protein A from Staphylococcus aureus. J. ImmunoL Methods 15, 57-66. CARROLL, R. B., HAGER, L., and DULBECCO,R. (1974). Simian virus 40 T antigen binds to DNA. Proc Nat. Acad Sci. USA 71.3754-3757.
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CHANG, CH., LUBORSKY, S. W., and MORA, P. T. (1977). Tumour-specific transplantation antigen from SV40 transformed cells binds to DNA. Nature (London) 269,438-440. CHANG, C., MARTIN, R. G., LIVINGSTON, D. M., LuBORSKY, S. W., Hu, C.-P., and MORA, P. T. (1979). Relationship between T-antigen and tumor-specific transplantation antigen in simian virus 40transformed cells. J. ViroL 29, 69-75. DEFENDI, V. (1963). Effects of SV40 virus immunization on growth of transplantable SV40 and polyoma virus tymors in hamsters. Proc. Sot. Exp. BtiL h&d 113,12-16. DENHARDT, D. T., and CRAWFORD,L. V. (1986). Simian virus 40 T-antigen: Identification of tryptic peptides in the C-terminal region and definition of the reading frame. J. V&l 34,315-329. DEPPERT, W., and HENNING, R. (1979). SV40 T-antigen related molecules on the surface of adenovirus-2-SV40 hybrid-virus infected HeLa cells and on SV40-transformed cells. Cold &n-&g Harbor Symp. Quant BioL 44.225-234. DEPPERT, W., HANKE, K., and HENNING, R. (1980). Simian virus 40 T-antigen-related cell surface antigen: Serological demonstration on simian virus 40-transformed monolayer cells in situ. J. ViroL 35, 505-518. DEPPERT, W. (1980). SV40 T-antigen related surface antigen: Correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant. %-o&y 104, 497-501. FIERS, W., CONTRERAS,R., HAEGEMAN, G., ROGIERS, R., VAN DE VOORDE, A., VAN HEWERSWYN, H., VAN HERREWEGHE, J., VOLCKAERT, G., and YSEBAERT, M. (1978) Complete nucleotide sequence of SV40 DNA. Nature (London) 273, 113-120. GOODING, L. R. (1977). Specificities of killing by cytotoxic lymphocytes generated in viva and in vitro to syngeneic SV40-transformed cells. J. Immund 118, 920-927. HABEL, K., and EDDY, B. E. (1963). Specificity of resistance to tumor challenge of polyoma and SV40 virus-immune hamsters. Proc. SW. Ezp. BioL &fed 113, 1-4. HENNING,
R., LANGE-MUTSCHLER,
J., and
DEPPERT,
W. (1981). SV40-transformed cells express SV40 Tantigen-related antigens on the cell surface. Virology 108,325-337. HUNTER, W. M., and GREENWOOD,F. C. (1962). Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194, 495-496. KHERA, K. S., ASHKENAZI, A., RAPP, F., and MELNICK, J. L. (1963). Immunity in hamsters to cells transformed in vitro and in viva by SV40. Tests for antigenic relationship among the papova viruses. Immuno&v 91, 604-613. KNOWLES, B. B., KONCAR, M., PFIZENMAIER, K., SOL-
CELL SURFACE BINDING TER, D., ADEN, D. P. and TRINCHIERI, G. (1979). Genetic control of the cytotoxic T cell response to SV40 tumor-associated specific antigen. J. Immund 122, 1798-1806. KOCH, M. A., and SABIN, A. B. (1963). Specificity of virus-induced resistance to transplantation of polyoma and SV40 tumors in adult hamsters. Proc Sot. Exp. Biol Med. 113,4-12. KRESS, M., MAY, E., CASSINGENA, R., and MAY, P. (1979). SVdO-transformed cells express new species of proteins precipitable by antiSV40 tumor serum. J. ViroL 31, 472-483. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680+X2. LANGE-MUTSCHLER, J., DEPPERT, W., HANKE, K., and HENNING, R. (1981). Detection of simian virus 40 T-antigen-related antigens by an ‘?-protein A binding assay and by immunofluorescence microscopy on the surface of SV40-transformed monolayer cells. J. Gen Viral 52, 301-312. LOWRY, 0. H., ROSEBROLJGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. BioL Chem 193, 265275. MARK, D. F., and BERG, P. (1979). A third splice site in SV40 early mRNA. Cold Spring Harbor Symp. &ant.
Biol.
44, 55-62.
POPE, J. H., and ROWE, W. P. (1964). Detection of specific antigen in SV40-transformed cells by immunofluorescence. J. Exp. Med. 120, 121-128. PRIVES, C., GILBOA, E., REVEL, M., and WINOCOUR, E. (1977). Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen. Proc. Nat Acad Sti USA 74, 457-461. REDDY, V. B., THIMMAPPAYA, B., DHAR, R., SUBRAMANIAN, K. N., ZAIN, B. S., PAN, J., CHOSH, P. K., CELMA, M. L., and WEISSMAN, S. M. (1978). The genome of simian virus 40. S&ence 200,494-501. ROGERS, M. J., LAW, L. W., and APELLA, E. (1977).
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Subcellular distribution of the tumor-specific transplantation antigen of SV40-transformed cells. J. Nat. Cancer Inst. 59,1291-X95. SCHMITT-VERHULST, A.-M., PETTINELLY, C. B., HENKART, P. A., LUNNEY, J. K., and SHEARER, G. M. (1978). H-2-restricted cytotoxic effecters generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins. J. Exp. Med 147, 352-368. SOULE, H. R., and BUTEL, J. S. (1979). Subcellular localization of simian virus 40 large tumor antigen. J Viral
30, 523-532.
SOULE, H. R., LANFORD, R. E., and BUTEL, J. S. (1980). Antigenic and immunogenic characteristics of nuclear and membrane-associated SV40 tumor antigen. J. ViroL 33, 887-901. TEVETHIA, S. S., BLASECKI, J. W., WANECK, G., and GOLDSTEIN, A. L. (1974). Requirement of thymus derived &positive lymphocytes for rejection of DNA virus (SV40) tumors in mice. J. Immunol 113, 1417-1423. TEVETHIA, M. J., and TEVETHIA, S. S. (1977). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). III. Involvement of SV40 gene A in the expression of TrAg in permissive cells. Virology 81, 212-223. TEVETHIA, S. S., PLYER, D. C., and TJIAN, R. (1980). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40-transplantation immunity in mice by purified SV40 T-antigen (D2 protein). Virology 107,1323. TOOZE,J. (1980). “DNA Tumor Viruses,” 2nd ed., Vol. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. TRINCHIERI, G., ADEN, D. P., and KNOWLES, B. B. (1976). Cell mediated cytotoxicity to SV40-specific tumor associated antigens. Nature (London) 261, 312-314. WICKNER, W. (1980). Assembly of proteins into membranes. Stie-nee 219,861-868.
117.173-185
(1982)
Cell Surface Binding Affinity of Simian Virus 40 T-Antigen JUTTA Department
of
LANGE-MUTSCHLER
Biochemistry,
AND
ROLAND
HENNING’
University of Ulm, P.O. Box .@66,0-7900 Ulm, Federal Republic of Germnnv Received July 23, 1981; accepted, October 20, 1981
Simian virus IO-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in wivo by the rejection of tumorigenic SV40transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SVQO-infected and -transformed cells express SV40 large tumor antigen (T-Ag) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 T-Ag solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of T-Ag to the cell surface (called “externally hound T-Ag”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive ‘%I-protein A assay using rabbit antisera directed against purified SV40 T-Ag. Serological analysis of T-Ag in cellular extracts and of externally bound T-Ag revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 T-Ag. We interpret our results as an indication that this property enables purified T-Ag to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.
INTRODUCTION
Simian virus 40 (SV40)-transformed cells express a SV40-specific tumor transplantation antigen (TSTA) encoded by the early region of SV40 DNA (for review see Tooze, 1980). TSTA is defined by cellular immune response demonstrated in vivo by the transplantation rejection test (Defendi, 1963; Khera et al., 1963; Habel and Eddy, 1963; Koch and Sabin, 1965) and in vitro by a T lymphocyte-mediated lysis of SV40-transformed cells (Tevethia et al., 1974; Tevethia and Tevethia, 1977; Trinchieri et al., 1976; Gooding, 1977; Knowles et al., 1979). The proteins known to be encoded by the early region of SV40 are large tumor antigen (T-Ag) which is located in the nucleus (Pope and Rowe, 1964) and small tumor antigen (t-antigen) which is found mainly in the cytoplasm (Prives et al., 1977). Several efforts to relate early SV40 pro’ Author dressed.
to whom
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requests
should
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teins to TSTA suggested that T-Ag or closely related antigens occur on the cell surface: (i) Using antisera directed against purified T-Ag, the presence of T-Ag or closely related antigens was demonstrated serologically on the surface of SV40-transformed cells (Deppert and Henning, 1979; Soule and Butel, 1979; Deppert et al., 1980; Soule et al., 1980; Henning et al., 1981; Lange-Mutschler et al., 1981). (ii) Biochemical analysis by cell surface ‘%I iodination using lactoperoxidase (Deppert and Henning, 1979) and of plasma membranes isolated from [35S]methionine-labeled SV40-transformed cells (Soule et al., 1980) demonstrated a T-Ag-like molecule with an identical electrophoretic migration behavior (MW = 94,000). (iii) Attempts using tsA mutants of SV40 failed to distinguish genetically between TSTA and nuclear T-Ag (Anderson et al., 1977), or between surface T and nuclear T-Ag (Deppert, 1980). (iv) The best evidence for the correlation between T-Ag and TSTA comes from the use of purified T-Ag which 0042~6822/82/030173-13$02.00/O Copyright 0 1922 by Academic Press, Inc. All rights of reproduction in any form reserved.
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immunized mice against a SV40 tumor cell challenge (Chang et al., 19’77; Chang et al., 1979); in addition, immunization with purified D2, a T-Ag-related protein encoded by adeno 2-SV40 hybrid virus (Ad2+D2) protected mice against a challenge of syngeneic SV40 tumorigenic cells and induced in vivo the generation of cytotoxic T lymphocytes sensitized against SV$O-transformed cells as demonstrated in vitro by a cytotoxicity assay (Tevethia et al., 1980). It thus seems to be clear that large SV40 T-Ag possesses TSTA activity in regard to the induction of the cellular immune response in vivo and possibly to the formation of TSTA as a cell surface target antigen for cytotoxic T lymphocytes. At present, all these observations are compatible with the hypothesis that T-Ag is pleiotropic, i.e., multifunctional T-Ag seems to occur simultaneously in nuclei and on the cell surface. Yet, the obvious biological and biochemical questions concerning this unique property of T-Ag remained unanswered. The facts, however, that immunization with T-Ag solubilized and purified to homogeneity protected mice against the SV40 tumor challenge suggested a new and special property of T-Ag in terms of cell surface binding affinity. Therefore, we investigated the binding affinity of T-Ag to the cell surface of SV40-transformed and various other cell lines. In the present study, we provide evidence that T-Ag solubilized from SV40transformed or -infected cells binds in significant amounts to the surface of living monolayer cells. MATERIALS
AND
METHODS
Cell Lines and Vim~s Mouse Balb/c fibroblasts (3T3) and the corresponding SV40-transformed cells (SV3T3, SVTZ), the naturally transformed human epithelial cell line (HeLa) and the SV40-transformed human fibroblasts (SVSO), the hamster embryonal cell line (BHK) and the SV40-transformed hamster fibroblasts (H 65190 B, kindly provided by Dr. V. Defendi, New York Uni-
AND
HENNING
versity, New York), were grown in petri dishes or in Roux bottles in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 510% calf serum. African green monkey kidney cells (TC-7) were grown in DMEM supplemented with 5% fetal calf serum. Confluent TC-7 cells were infected with SV40 virus strain 777 (approximately 10 PFU/cell) for 24 or 48 hr. Sera (i) Rabbit anti-T-Ag serum. T-Ag extracted from SW0 cells and subsequently purified by immunoprecipitation and SDSpolyacrylamide gel electrophoresis was used for immunization of rabbits as described previously (Deppert and Henning, 1979). The titer against nuclear T-Ag of the rabbit anti-T-Ag serum used in the present study (No. 20) was determined by indirect immunofluorescence microscopy according to Pope and Rowe (1964) on ethanol-fixed SVT2 cells (T-Ag titer 1:3200). The titer against surface T was determined by the ‘251-protein A assay on living SVT2 cells (1:20). In order to abolish an unspecific binding activity of the rabbit anti-T-Ag serum No. 20 to the cell surface of living cells, this serum was absorbed once on ethanol-fixed 3T3 cells as described previously (Henning et al., 1981). The other sera were used without absorption. (ii) Hamster SVIO tumor serum. Hamster SV40 tumor sera were obtained from inbred C’Lak hamsters 3-6 weeks after subcutaneous injection with 2 X lo6 H 65/ 90 B cells as described previously (Henning et al., 1981). After screening for similarly high titers against nuclear T-Ag as determined by indirect immunofluorescence according to Pope and Rowe (1964) on ethanol-fixed SVT2 cells hamster SV40 tumor sera were pooled (hamster SV40 tumor serum pool 3, titer 1:lOOO). This pool had a low titer against surface T (1:lO) as determined by the ‘%I-protein A assay on living SVT2 cells. (iii) Rabbit anti-actin serum. Rabbit anti-actin serum was raised in rabbits immunized with purified actin of chicken
CELL
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T-ANTIGEN
175
Lange-Mutschler et al., 1981). Thirty micrograms of protein A (Pharmacia) was labeled for 10 min at room temperature with 1 mCi Nal? (NEN) in 0.5 ml PBS by the chloramine-T method according to Hunter and Greenwood (1962). ‘%I-Labeled protein A was purified by gel chromatography in PBS on Sephadex G-25. The specific radioactivities varied between 2 X 10’ and 4 X 10’ cpm/pg. Cells (2 X 103) harvested from monolayer cultures were seeded per well in Terasaki microtiter plates and kept overnight at 37” to become adherent. To study the cell surface binding activity of T-Ag 5 ~1 of cell extract corresponding to l-2 X lo5 cells was added to each well for 1 hr at 37”. In a separate set Cell Extracts of experiments we observed that the steady Confluent monolayer cells grown in Roux state of the uptake of T-Ag onto the surbottles or petri dishes were harvested me- face of living cells was achieved within 60 chanically by scraping the cells off using min. After removal of the extracts the a rubber policeman. After washing the plates were washed extensively by gentle cells three times with HEPES-buffered shaking in HEPES-DMEM over a period Dulbecco’s modified Eagle’s medium of 45 min including 12 changes of washing (HEPES-DMEM, pH 7.3) 2 X 10’ cells buffer. The wells were then incubated with (SVSO, HeLa, SV3T3, 3T3, H 65190 B, 5 ~1 of serum diluted 1:lO in HEPESBHK) or 4 X lo7 cells (SVTB) were sus- DMEM for 1 hr at 37”. After another sepended in 1 ml HEPES-DMEM. In order ries of washes the cells were incubated to inhibit the activity of proteases 100 ~1 with ‘%I-labeled protein A for 30 min at of Trasylol (Bayer) was added per one room temperature. Finally, the plates were milliliter of cell suspension. Cells were frowashed over a period of at least 2 hr with zen at -70” and kept in this form up to several changes of HEPES-DMEM until 3 months. Extracts of frozen cells were the background binding of lzjI-protein A prepared freshly for every experiment. to empty wells was reduced to approxiAfter thawing, the cell suspensions were mately 100 cpm. The wells were dried, cut homogenized at 4’ with a tightly fitting out of the plates, and counted in a gamma Dounce homogenizer. To assure that cells counter. Each set of Terasaki plates has were completely homogenized the suspento be examined for background binding sions were sonicated once at 4” for 30 set properties in the absence of cells in order and checked microscopically for the ab- to rule out an unspecific binding of extract sence of intact cells or nuclei. Cellular de- proteins, immunoglobulins, or ‘%I-protein bris and subcellular particles were re- A to the plastic surface. In the case of moved by ultracentrifugation at 105,000 unspecific binding it could be considerably g for 30 min at 4”. In the following the reduced (lo-20% of the binding to cells) supernatants are called “cell extracts.” by precoating the plates with 15% human serum albumin (HSA) solution overnight and by addition of 15% HSA to cell exlz51-Protein A Assay tracts, antisera, and ‘%I-protein A soluThe ‘251-protein A assay (Brown et al., tion. 1977) performed in the present study on As demonstrated by immunofluoresuntreated monolayer cells was modified as cence microscopy the cells remained viable described previously (Henning et al., 1981; and intact during the ‘?-protein A test.
breast muscles as described previously (Arnold et al., 1968; Henning et al., 1981). The titer against actin was determined by indirect immunofluorescence microscopy on ethanol-fixed 3T3 cells (titer 1:lOO). (iv) Rabbit anti-cell serum. Rabbit anticell serum was obtained from rabbits immunized intraperitoneally three times at 3-week intervals with 10’ H 65/90 B cells. This xenoantiserum (No. 15) has high titers as determined by indirect immunofluorescence microscopy on living ceils against cell surface antigens of various cell lines (H 65/90 B, SV80,3T3) (ranging between 1:3000 and 1:6000).
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LANGE-MUTSCHLER
Using SV40-transformed cells and rabbit anti-T-Ag serum it could be shown that no more than 0.05% of the cells (i.e., one or two dead cells per well) became positive for nuclear T-Ag. Radiolabeled Cell Extracts Approximately 2 X lo6 SW30 cells grown in one petri dish (lo-cm diameter) were radiolabeled by incubation with 1 ml of methionine-free DMEM containing 40 &i of $S]methionine (Amersham) for 4 hr at 37”. After washing the cells were frozen and thawed once. Extracts of labeled cells (2 X 10’ cells/ml PBS) were prepared after homogenization at pH 7.3 as described above. The protein concentration in the cell extracts was determined by the method of Lowry (1951). In order to determine the specific radioactivity of the proteins, aliquots of the extracts were precipitated with ice-cold trichloroacetic acid (TCA, final concentration 10%) w/v), washed several times with ethanol at -2O”, dried, dissolved and counted in 10 ml of scintillation liquid (Quickszint, Zinsser, Frankfurt FRG). The specific radioactivity (cpm/ pg protein) of total protein in a representative experiment using radiolabeled extracts of SW30 cells (7 mg protein/ml) was 5 X lo5 cpm/mg. Immunoprecipitation Electrophoreti
and SDS-Gel
To check the T-Ag specificity for cell surface binding experiments 300 ~1 of extracts was incubated with 10 ~1 of hamster SV40 tumor serum, or with 10 ~1 of rabbit anti-T-Ag serum or with the corresponding normal sera. After incubation for 1 hr at 4” 100 ~1 of pelleted protein A-Sepharose (Pharmacia) preswollen in phosphatebuffered saline (PBS, pH 7.3) was added. After shaking gently for 1 hr at 4” the immunoprecipitates were pelleted by centrifugation. The supernatants were used to determine the cell surface binding affinity of T-Ag by the 12SI-protein A assay on SV3T3 and 3T3 cells. The immunoprecipitates were analyzed for T-Ag by SDSpolyacrylamide gel electrophoresis (SDSPAGE). After extensive washing with
AND
HENNING
NET buffer (0.15 M NaCl, 5 mM EDTA, 50 mM Tris-HCl, 0.5% NP40, pH 7.4) and finally with 50 mM NH4HC03, the immune complexes were eluted from the protein ASepharose using 500 ~1 of elution buffer (50 mM NH4HC03, 2% SDS, 5% 2-mercaptoethanol). The eluates were lyophilized, dissolved in 20 ~1 sample buffer (0.1 M Tris-HCl, pH 6.8, 5% 2-mercaptoethanol, 0.005% bromophenol blue) and then run on 12% SDS-polyacrylamide slab gels at a constant current of 12 mA (Laemmli, 1970). Proteins were visualized by staining with Coomassie blue. For the determination of the content of radiolabeled T-Ag [?S]methionine-labeled extracts of 2 X lo6 SV80 cells were immunoprecipitated with hamster SV40 tumor serum as described above. SDS-PAGE was performed on lo-cm 12% polyacrylamide gels in glass tubes (6-mm diameter) using a 1.5-cm stacking gel containing 5% acrylamide at a constant current of 3 mA/ tube at room temperature. The gels were frozen immediately at -2O”, cut into 2-mm slices, eluted overnight in 200 ~1 water, and counted for YS cpm in 10 ml scintillation liquid (Quickszint, Zinsser). Comparison of the ?S cpm of T-Ag and the specific radioactivity of the total cellular proteins (?S cpm/pg) after a 4-hr labeling period determined in the TCA precipitates revealed a T-Ag content of approximately 1.4 pg/ml equivalent to 0.1% of the total protein amount. This estimation was based on the average of specific radioactivity of total cellular proteins. RESULTS
Solubilixed SV40 T-Antigen Binds to the Cell Sqfkce of Various Cell Lines It was possible to demonstrate surface T on living SV40-transformed cells by immunofluorescence microscopy and by the ‘%I-protein A assay using sera from rabbits immunized with purified T-Ag and sera from SV40 tumor-bearing hamsters (Henning et al., 1981). As expected, in the present study incubation of three SV40transformed cell lines derived from three species (human SV80 cells, mouse SV3T3 cells, hamster H 65/90 B cells) with the
CELL
SURFACE
BINDING
rabbit anti-T-Ag serum resulted in a low but significant binding of ‘%I-protein A molecules (Table 1) confirming the presence of surface T on SV40-transformed monolayer cells in situ. The incubation of monolayer cells in situ with T-Ag-containing extracts from SV40-transformed cells led to a significant increase in the amount of anti-T-Ag antibodies and ‘*I-protein A bound to the surfaces of all cell lines tested by a factor ranging between 2 and 5 (P < 0.0005) (Table 1). In contrast, extracts from the naturally transformed human cell line (HeLa) did not alter the number of anti-T-Ag antibody binding sites on the surface of all three SV40-transformed cells tested (Table 1; P < 0.025). Thus, the number of surface T-like molecules on SV40-transformed cells was only increased by the external addition of surface binding T-Ag molecules (called externally bound T-Ag) from extracts of SV40-transformed cells, but not by an increase in the serological accessibility of original surface T. These observations were confirmed using extracts from 3T3 or BHK cells (data not shown). The T-Ag specificity of the anti-T-Ag serum binding activity is demonstrated by the subtraction of data obtained with normal rabbit serum from those obtained with rabbit anti-T-Ag serum. These results are proved statistically by Student’s t test. As outlined under Materials and Methods, after performing the ‘l-protein A assay the wells were routinely checked by immunofluorescence microscopy for the presence of nuclear TAg positive cells. Here, it should be pointed out that this examination of living monolayer cells for nuclear T-Ag was particularly important after incubation with cell extracts. After performing the extensive and careful washing procedure we could not detect more than 0.05% T-Ag-positive cells per well since dead cells disappeared during the washing steps. Additionally, the viability of target cells demonstrated by the impermeability for antibodies was assessed by using a rabbit antiactin serum under identical conditions, i.e., after incubation of living target cells with extracts (see Table 4). We can assume that the increase in the number of T-Ag bind-
OF SV40
T-ANTIGEN
177
ing sites corresponds to externally bound T-Ag. Thus, solubilized T-Ag in cell extracts has a cell surface binding affinity on SV40-transformed cells. The term affinity is used in a general sense to apply to binding between soluble T-Ag or T-Ag assembled with another component and binding sites on the cell surface. The presence of surface T on SV40transformed cells raised the question of possible binding between solubilized T-Ag and surface T as a putative receptor due to the occurrence of aggregates between native T-Ag molecules in solution (Carroll et al., 1974). Therefore, we extended these experiments using naturally transformed human (HeLa) and embryonic mouse and hamster cell lines (3T3, BHK) as target cells which do not expose T-Ag or serologically related surface antigens. As expected, all three cell lines were negative for surface T (Table 2). Again, incubation of these target cells with extracts from SV40-transformed cells of three different species (SVSO, SV3T3, H 65/90 B) led to a number of externally bound T-Ag molecules comparable with the number of surface T molecules on SV40-transformed cells as shown in Table 1. These data show that solubilized T-Ag does not need natural surface T present on SV40-transformed cells in order to bind to the cell surface. In conclusion, both sets of results shown in Tables 1 and 2 suggest a general cell surface binding affinity of soluble TAg or at least of certain T-Ag subclasses. A comparison of the data shown in Tables 1 and 2 suggests a stronger cell surface binding affinity of T-Ag to SV40transformed cells than to other cell lines. In order to test the possibility of a saturable interaction, we analyzed the cell surface binding affinity of T-Ag under identical conditions by varying the concentration of cellular extracts. Figure 1 shows that the increase of T-Ag-related molecules bound to the cell surface of SV3T3 cells reached an apparent saturation only at high concentrations, whereas on 3T3 cells comparable results were not yet obtained at these concentrations. Comparative analysis of the stability of externally bound T-Ag and surface T indicated
178
LANGE-MUTSCHLER
AND
TABLE
HENNING
1
'?-PROTEIN A ASSAY FOR THE BINDING AFFINITY 0~ T-Ag SOLUBUJZED FROM SV&TBANSFORMBD TOTHE CELLSURFACEOFSV~O-TRANSFORMEDMONOLAYERCELLS ‘“I-Protein
A molecules X lo-’ bound per microtiter
plate well
After incubation with cell extracts from different transformed cell lines Antisera Anti-T-Ag serum Normal rabbit serum
Target cells SVCQ
T-Ag specific binding Anti-T-Ag serum Normal rabbit serum
SV3T3
T-Ag specific binding Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
H 65/SO B
CELLS
SV40-
After incubation with DMEM
SVSO
sv3T3
984*34 532220
2571 f 337 640 + 108
24m1tm 434 2 101
1685 + 192 647 rt 113
1038 + 145 480+ 38
452 + 39
1931 + 353
1595 + 278
1038 * 222
558 + 149
327 k 24 141 + 23
1188 + 134 176 + 35
1252 * 174 314 i 42
1432 + 167 236+ 63
461 + 59 264k 62
186 + 33
1012 + 197
938 t 178
1136 r 178
193+
405 + 54 130 f 12
1274 r 310 ill? 17
780 f 103 139+ 28
880 r 122 149 t 21
n.d. n.d.
275655
1163 + 317
6415 106
731 + 123
n.d.
HG&OB
HeLa
85
Note. SVQO-transformed target cells (human SWO, mouse SV3T3, hamster H 65/3O B cells) were incubated for 1 hr at 37” with T-Ag-containing extracts prepared from SVIO-transformed cells (SVSO. SV3T3. H 65/9O B). Control experiments were performed witb an extract from HeLa cells or only with HEPES-buffered DMEM. After washing. the cells were incubated with 1:lO dilutions of rabbit anti-T-Ag- of normal rabbit serum in HEPES-buffered DMEM and finally with ‘“I-protein A as described under Materials and Methods. The results represent the ‘“I-protein A molecules X lo-’ (mean + SD, N = 10) per one Terasaki microtiter plate well seeded with 2 X lo’ cells. The T-Ag specific binding indicating surface T and externally bound T-Ag was calculated by the formula: IS - NS f (SD IS* + SD NSP)1/2, IS is the number of protein A molecules bound to cells incubated with rabbit anti-T-Ag serum; NS is the number of protein A molecules bound to cells incubated with normal rabbit SW”lll
that both types of antigens stayed stable up to 24 hr after precoating with anti-TAg sera (data not shown). Recently it was reported, that T-Ag expressed in SV40-transformed cells exists in various species having different biochemical properties when compared with T-Ag expressed in SV40-infected monkey kidney cells (Kress et al., 1979). We therefore tested whether T-Ag solubilized from SV40-infected TC-‘7 cells express a cell surface binding affinity comparable to that of T-Ag extracted from SV40-transformed cell lines. As shown in Table 3, T-Ag extracted from TC-‘7 cells SV40 infected for 24 or 48 hr showed a cell surface binding affinity to mouse 3T3 cells similar to T-Ag from SV40-transformed cells. In control experiments, extracts from noninfected TC-7 cells did not alter the binding of antiT-Ag antibodies to the cell surface of 3T3 cells. As expected from the increase of the production of T-Ag the amount of externally bound T-Ag obtained 48 hr p.i. increased significantly when compared with the binding data determined 24 hr p.i.
Similar results were obtained using TC-7 cells as target cells for extract incubation (data not shown). These data clearly demonstrate, that there was no distinct difference in the cell surface binding affinities between T-Ag extracted from SV40transformed and from lytically SV40-infected cells. Externally Bound and Soluble T-Ag Molecules Are Serologicall~ Similar
The data presented so far strongly suggest that the increase of surface T molecules on cells incubated with extracts from SV40-transformed or -infected cells is due rather to the binding of T-Ag or closely related SV40-specific molecules than to the binding of serologically cross-reacting but cellular proteins which are expressed merely in SV40-transformed cells. This interpretation was checked by examining whether cell extracts were still able to increase the number of surface T molecules after specific removal of T-Ag by immunoprecipitation. To extend the serological
CELL SURFACE
BINDING
179
OF SV40 T-ANTIGEN
TABLE 2 ‘=I-PROTEIN A ASSAY FOR THE BINDING AFFINITY OF T-Ag SOLUBILIZED FROM SV40-TRANSFORMED CELLS TO THE CELL SURFACE OF HeLa, 3T3 AND BHK CELLS ‘?-Protein
A molecules X 10m6bound per microtiter
well
After incubation with cell extracts from different SV40-transformed cell lines Antisera
Target cells
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
HeLa
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
3T3
Anti-T-Ag serum Normal rabbit serum T-Ag specific binding
BHK
After incubation with DMEM 352 1- 54 + 32 97 f 62
255
SV80
H 65/90 B
SV3T3
993 -t 234 f 56 667 f 240 326
736 f 112
938 rf: 164
422f
46
432k
314
Lk 121
506
67
?I 177
174 + 22 50 f 49
935 + 143 194 + 36 741 + 147
603 150
+ 81 f 18 453 f 83
361f 61+ 300
f
29
140 f 27 103 + 20 37 zk 33
620 f 150 * 470 *
644k 42 279 f 33 365 + 53
538 277
f f
22
224f44
87
30 92
261+
26 12
91 94
Note. Human HeLa, mouse 3T3, hamster BHK cells were incubated with T-Ag containing extracts prepared from different SV40-transformed cell lines (SVSO, SV3T3, H 65190 B) or with HEPES-buffered DMEM as control. T-Ag molecules externally bound to the cell surfaces were determined by incubation with rabbit anti-T-Ag serum or normal rabbit serum and ‘?-protein A. The results represent the number of ‘%I-protein A molecules X 10m6(mean + SD, N = 10) bound per one Terasaki microtiter plate well seeded with 2 X lo3 cells. The T-Ag specific binding was calculated in the same way as for Table 1.
specificity of this type of experiment we used two different types of anti-T-Ag sera: hamster SV40 tumor serum (HaT, pool No. 3) for immunoprecipitation of T-Ag from cell extracts and rabbit anti-T-Ag serum for the measurement of externally bound T-Ag. A representative experiment is shown in Fig. 2: Aliquots of an extract of SW30 cells were immunoprecipitated with either hamster SV40 tumor serum or normal hamster serum (NHS). The immunoprecipitates were analyzed by SDSPAGE, and, as expected, hamster SV40 tumor sera precipitated T-Ag while NHS did not recognize T-Ag (Fig. 2B). The corresponding supernatants of both immunoprecipitates were then applied for the cell surface binding assay on SV3T3 cells under standard conditions using rabbit anti-T-Ag serum. Figure 2A shows that the typical increase in the binding of antiT-Ag serum on SV3T3 cells after incubation with SW30 cell extract preprecipitated with normal hamster serum was not al-
tered when compared with SV3T3 cells incubated with the untreated extract. In contrast, incubation of SV3T3 cells with SW30 cell extract preprecipitated with hamster SV40 tumor serum did not lead to an increase in the T-Ag specific binding when compared with cells incubated merely with medium. Similar results were obtained using 3T3 as target cells for a TAg cell surface binding assay (data not shown). These results confirm the data presented in Tables l-3 and moreover, they allow two conclusions in regard to the specificity of the cell surface binding affinity of T-Ag molecules from cell extracts: (i) removal of T-Ag from cell extracts by immunoprecipitation was clearly correlated with the disappearance of the cell surface binding affinity of T-Ag; (ii) using hamster SV40 tumor serum for the immunoprecipitation of T-Ag and rabbit anti-T-Ag serum for the determination of externally bound T-Ag molecules indi-
180
LANGE-MUTSCHLER
without extract
extract
dilution
FIG. 1. Binding of T-Ag to 3T3 cells and SV40transformed monolayer cells (SV3T3) at different concentrations of SV3T3 cell extracts. Mouse fibroblasts (3T3) and the corresponding SV40-transformed cells (SV3T3) were incubated with different dilutions of an extract prepared from SV3T3 cells. The concentration of undiluted extract (2 X lo7 SV3T3 cells/ml) was the same as used for the experiments described in Tables l-3. T-Ag molecules present on the cell surfaces were detected using 1:lO dilutions of rabbit anti-T-Ag serum or normal rabbit serum and ‘251-protein A. The data represent the T-Ag specific binding calculated in the same way as for Table 1 (N = 6).
cated the serological cross-reaction of both types of antisera. This situation corresponds exactly to the previously described cross-reacting specificities of both anti-TAg antibodies used for the demonstration of surface T on SV40-transformed cells (Henning et al., 1981). The observation of the cell surface binding affinity of T-Ag in the presence of total cell extracts prompted the obvious question as to whether other cellular proteins might also bind in comparable amounts to the surface of monolayer cells in situ. Although, due to the reasons mentioned above, there seems to be no doubt about the cell surface binding affinity of T-Ag, other proteins might influence the binding affinity of T-Ag. Due to the extensive rinsing procedure (see Materials and Methods) designed to wash off cellular proteins and simultaneously to maintain monolayer target cells adherent, presumably several cellular proteins present in the cell ex-
AND
HENNING
tracts might bind to the surface of target cells. Therefore, we examined the binding of cellular proteins by three approaches. (i) The amount of proteins bound to the surface of target cells was estimated using radiolabeled extracts of cells. In a representative experiment we determined the specific radioactivity of total cellular proteins in [?S]methionine-labeled SW30 cell extracts (5 X lo5 ?S cpm/mg). The incubation of SV80 target cells with these extracts (1.8 X lo4 ?S cpm/well corresponding to 35 pg protein/well) did not lead to a measurable amount of $S]methioninelabeled protein bound to the target cells (20 f 17 cpm/well). This result indicated that no cellular protein bound to the cell surfaces was detectable under these experimental conditions. (ii) Binding properties of various proteins to the cell surface were estimated using polyspecific rabbit antisera directed against hamster cells (H 65/90 B). This antiserum has a high titer against cell surface antigens on several cell lines (see Materials and Methods). A considerable number of binding sites of these- polyspecific antibodies was detected on untreated SVT2 cells but no significant increase was observed after incubation of these cells with extracts of the same cells (P < 0.475) (Table 4). (iii) The cell surface binding affinity of actin as a good example of a major intracellular protein was determined by using a rabbit antiactin serum. The data presented in Table 4 show that in comparison with cells incubated with medium, no significant amounts of both types of antibodies were bound to the cell surface after incubation of target cells with cell extracts. In summary, despite the sensitivity of the assay and the specificity of the antisera used none of these data indicate a significant increase in ceil surface binding of cellular proteins after incubation of living target cells with extracts. DISCUSSION
Simian virus 40 T-Ag appears to bind in significant amounts to the cell surface of various transformed and nontransformed cell lines. This finding helps to explain the mechanism by which immuni-
CELL SURFACE BINDING
181
OF SV40 T-ANTIGEN
TABLE 3 ‘z-I-P~~~~~~
A ASSAY FOR THE BINDING AFFINITY OF T-Ag SOLUBILIZED FROMSV40-INFECTED TC-7 CELLS TOTHE CELLSURFACEOF~T~ CELLS ‘?-Protein
Antisera Anti-T-Ag serum Normal serum T-Ag specific binding
3T3 cells incubated with DMEM
A molecules X 10m6bound per microtiter
plate well
3T3 cells incubated with extract of TC7 cells
3T3 cells incubated with extract of SV40-infected TC-7 cells (24 hr p.i.)
3T3 cells incubated with extract of SV40-infected TC-7 cells (48 hr p.i.)
194 f 52 170 Ik 45 24 + 69
433 f 75 190 f 29 243 f 80
947 + 252 248 f 48 699 + 256
248+40 252 + 72 -4 + 82
Note. Mouse 3T3 cells were incubated with T-Ag-containing extracts prepared from TC-‘7 cells infected with SV40 virus for 24 or 48 hr. As a control, 3T3 cells were incubated with uninfected TC-7 cells or only with growth medium. T-Ag molecules externally bound to the cell surfaces were determined by incubation with rabbit anti-T-Ag serum or normal rabbit serum and ‘251-protein A. The results represent the number of ‘%I-protein A molecules X 10e6 (mean * .SD, N = 6) bound per one Terasaki microtiter plate well seeded with 2 X l@ cells. The T-Ag specific binding was calculated in the same way as for Table 1.
zation with purified T-Ag induces the rejection of SV40-induced tumors in mice. At the moment, we have no serological or biochemical data hinting at any significant differences between native surface T as detected on SV40-transformed cells in situ and externally bound T-Ag. Hence, the TSTA activity of highly purified T-Ag might be caused by its cell surface binding affinity. The evidence for cell surface binding affinity of T-Ag in extracts from SV40transformed cells is established by several qualitative and semiquantitative results: (i) T-Ag from SV40-transformed or -infected cells binds to different cell lines independent of the species and the cell transformation. (ii) Removal of T-Ag from cell extracts by immunoprecipitation led to an abolishment of its cell surface binding activity. Using two different types of anti-T-Ag sera demonstrates the serological similarity between T-Ag immunoprecipitated from all extracts and externally bound T-Ag. (iii) Extracts from non-SV40transformed cells do not alter the amount of surface T on SV40-transformed cells. Based on these observations we have to reconsider possible differences and simi-
larities between surface T and nuclear TAg as discussed previously by ourselves and other laboratories. As pointed out in the Introduction, there is so far no genetic or biochemical evidence for molecular differences between both forms of T-Ag. Analyzing the presence of surface T on formaldehyde-fixed cells by immunofluorescence microscopy using SV40 tumor sera or rabbit anti-T-Ag sera, we previously described serological differences in comparison with nuclear T-Ag in cells fixed with formaldehyde followed by methanol/ acetone treatment (Deppert et al., 1980). This interpretation was principally not compatible with observations made on living cells. Soule et al. (1980) described unique antigenic determinants of surface T on living cells, but only after putting monolayer cells into suspension with EDTA or after storing these cells in suspension in 90% buffered glycerol at -30”. However, using an ‘%I-IgG-blocking assay showed that antisera specifically directed against surface T as well as SV40 tumor sera blocked the cell surface binding of rabbit antisera directed against purified and denatured T-Ag (Soule et al., 1980). Similarly, sera against purified T-Ag could
LANGE-MUTSCHLER
AND HENNING
B extract
+
extract
extract Drecbitated with HaT
HaT
NHS
NHS
25
FIG. 2. Cell surface binding activity of T-Ag in cell extracts peprecipitated with anti-T-Ag serum. Aliquots of extract prepared from SV30 cells were immunoprecipitated with undiluted hamster SV40 tumor serum (HaT) or with normal hamster serum (NHS) and protein A-Sepharose. (A) iz51Protein A assay. Untreated extract and the supernatants of the immunoprecipitates were analyzed by the ‘=I-protein A assay for the presence of cell surface binding T-Ag molecules: SV3T3 cells were incubated with untreated extract (+ extract), extract immunoprecipitated with hamster tumor serum, extract immunoprecipitated with normal hamster serum, or incubated with growth medium as a control (- extract). Externally bound T-Ag was determined using rabbit anti-T-Ag serum or normal rabbit serum and ‘?-protein A. The data represent the T-Ag specific binding (mean f SD, N = 6) calculated in the same way as for Table 1. (B) SDS-polyacrylamide gel electrophoretic analysis of unlabeled immunoprecipitates. The immunoprecipitates were analyzed on a 10% SDSpolyacrylamide slab gel. The proteins were stained with Coomassie blue.
be demonstrated to block the binding of hamster SV40 tumor sera to cell surfaces (Henning et al., 1981). These observations indicated directly the very similar properties of different antisera with respect to the specificity for surface T. At present, there is no direct evidence available for serological dissimilarities between surface T and nuclear T-Ag. It should be noticed that T-Ag extracted and enriched from nuclei isolated from SV40-transformed cells possesses a potent TSTA activity (Rogers et al., 1977). Both observations, the cell surface binding affinity of solubilized T-Ag and that surface T is serologically indistinguishable from nuclear T-Ag raise the question whether surface T occurs as a membraneassociated or -integrated protein. For the latter possibility one has to assume the
classical signal sequence mechanism (Blobe1 and Sabatini, 1971; Blobel and Dobberstein, 1975) or the trigger hypothesis suggesting an internal sequence-mediated membrane insertion (for review see Wickner, 1980). The amino acid sequence of TAg derived from the nucleotide sequence of SV40 DNA (Fiers et al., 1978; Reddy et al., 1978) does not seem to provide sufficiently extensive hydrophobic regions complying with the trigger hypothesis nor with the signal sequence. Alternatively, Mark and Berg (1979) reported a new splicing region in early SV40 RNA that would be able to code for a T-Ag with a hydrophobic carboxy terminus (T*-Ag). However, according to careful tryptic peptide analyses of T-Ag extracted from SV40 wild-type and dl 1263 mutant-infected cells an apolar region at the carboxy ter-
CELL SURFACE
BINDING TABLE
4
‘%I-protein
Antiserum Rabbit anti-cell serum Rabbit antiactin serum Normal rabbit serum
183
OF SV40 T-ANTIGEN
A molecules X 1O-6 bound per microtiter plate well
After incubation with DMEM 108,319 + 8091 131 + 11 117 * 45
After incubation with extract of SVT2 cells 112,642 _+ 6942 110 f 21 75f 15
Note. SV40-transformed monolayer cells (SVT2) were incubated with an extract prepared from SVT2 cells or with HEPES-buffered DMEM. Cellular proteins present on the cell surface were measured using polyspecific rabbit anti-cell serum and ‘%I-protein A. Actin molecules from cell extracts externally bound to the cell surface were assayed using antiactin serum and lz51-protein A. The data represent the number of protein A molecules X 1O-6 (mean + SD, N = 6) bound per one Terasaki microtiter plate well seeded with 2 X lo3 cells.
minal end was not detectable (Denhardt and Crawford, 1980). For these and other reasons requiring a variety of distinct biochemical properties for integral membrane proteins it seems to be unlikely that T-Ag or subclasses (surface T) belong to the family of integrated membrane proteins. Although evidence against regarding surface T as an integrated membrane protein remains to be determined, at the moment, for several reasons we favor considering surface T as a membrane-associated protein. First, both the present observations about the cell surface binding affinity and the induction of the TSTA activity by solubilized T-Ag are compatible with the idea that surface T is a membraneassociated protein. Second, the serologitally detectable similarity between surface T, externally bound T-Ag, and nuclear T-Ag indicates the possibility that surface T arises by leaking of T-Ag from living or dead cells. This idea is supported by the detection of soluble T-Ag in the growth medium of internally radiolabeled cells (umpublished observations). The question as to whether this mechanism causes the pleiotropic expression of T-Ag in nuclei and on the cell surface remains to be answered. According to our present knowledge our
findings can only suggest that T-Ag has a certain cell surface binding affinity without allowing us to define the type of binding sites. Since the binding experiments were performed by using extracts obtained from SV40-transformed and -infected cells but not with enriched or even highly purified T-Ag we do not know whether T-Ag or certain subclasses bind directly to unspecific binding sites, to putative receptors, or alternatively, whether T-Ag binds indirectly by using ligands such as nonviral T-Ag or nucleic acids. It should be noticed that the relatively high concentrations of cell extracts are necessary with respect to the total capacity of cell extracts to demonstrate the presence of externally bound T-Ag (see Fig. 1). One explanation of this event might be an methodologically intrinsic aspect: the total amount of T-Ag available for cell surface binding events might be considerably reduced during the preparation of cellular extracts under physiological conditions (i.e., ionic strength, pH, absence of denaturing agents) by removing the cellular debris including the plasma membranes with externally bound T-Ag by centrifugation. In interpreting our findings in terms of their possible immunological consequences it is striking to recognize that the
184
LANGE-MUTSCHLER
activity of highly purified and soluble TAg to stimulate a SV40-specific cellular immune response in mice can in fact be correlated with a cell surface binding property. It has recently been shown that incubation of spleen cells with soluble proteins such as trinitrophenylated bovine serum albumin or ovalbumin renders these cells susceptible to lysis by hapten-specific cytotoxic T lymphocytes (Schmitt-Verhulst et al., 1978; Ballas and Henney, 1979). It thus would be interesting to know whether the attachment of T-Ag, released from SV40-transformed cells, to normal cells might lead to the formation of a SV40 tumor-specific transplantation antigen. ACKNOWLEDGMENTS
The authors thank Ms. Cornelia Jenkner for technical assistance and Mrs. Madge B. Henning for the help with the manuscript. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (He 882/4 and He 882/5-3). REFERENCES ANDERSON, J. L., MARTIN, R. G., CHANG, Ch., MORA, P. T., and LIVINGSTON, D. M. (1977) Nuclear preparation of SV40 transformed cells contain tumor specific transplantation activity. Vidogg 76, 420425. ARNOLD, H., HENNING, R., and PETTE, D. (1968). Binding of glycolytic enzymes to structure proteins of the muscle. Eur. J. Biochem 6, 163-171. BALLAS, Z. K., and HENNEY, C. S. (1979). Generation of H-2 restricted cytotoxic T cells by trinitrophenylated proteins in vitro: Specificity and requirements. .I ImmunoL 123,1696-1704. BLOBEL, G., and SABATINI, D. D. (1971). Ribosomemembrane interaction in eucaryotic cells. Bimnembrunes 2,193-195. BLOBEL, G., and DOBBERSTEIN, B. (19’75). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. CelL BioL 67,852862. BROWN, J. P., KLITZMAN, J. M., and HELLSTR~~M, K. E. (1977). A microassay for antibody binding to tumor cell surface antigens using ‘%I-labelled protein A from Staphylococcus aureus. J. ImmunoL Methods 15, 57-66. CARROLL, R. B., HAGER, L., and DULBECCO,R. (1974). Simian virus 40 T antigen binds to DNA. Proc Nat. Acad Sci. USA 71.3754-3757.
AND
HENNING
CHANG, CH., LUBORSKY, S. W., and MORA, P. T. (1977). Tumour-specific transplantation antigen from SV40 transformed cells binds to DNA. Nature (London) 269,438-440. CHANG, C., MARTIN, R. G., LIVINGSTON, D. M., LuBORSKY, S. W., Hu, C.-P., and MORA, P. T. (1979). Relationship between T-antigen and tumor-specific transplantation antigen in simian virus 40transformed cells. J. ViroL 29, 69-75. DEFENDI, V. (1963). Effects of SV40 virus immunization on growth of transplantable SV40 and polyoma virus tymors in hamsters. Proc. Sot. Exp. BtiL h&d 113,12-16. DENHARDT, D. T., and CRAWFORD,L. V. (1986). Simian virus 40 T-antigen: Identification of tryptic peptides in the C-terminal region and definition of the reading frame. J. V&l 34,315-329. DEPPERT, W., and HENNING, R. (1979). SV40 T-antigen related molecules on the surface of adenovirus-2-SV40 hybrid-virus infected HeLa cells and on SV40-transformed cells. Cold &n-&g Harbor Symp. Quant BioL 44.225-234. DEPPERT, W., HANKE, K., and HENNING, R. (1980). Simian virus 40 T-antigen-related cell surface antigen: Serological demonstration on simian virus 40-transformed monolayer cells in situ. J. ViroL 35, 505-518. DEPPERT, W. (1980). SV40 T-antigen related surface antigen: Correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant. %-o&y 104, 497-501. FIERS, W., CONTRERAS,R., HAEGEMAN, G., ROGIERS, R., VAN DE VOORDE, A., VAN HEWERSWYN, H., VAN HERREWEGHE, J., VOLCKAERT, G., and YSEBAERT, M. (1978) Complete nucleotide sequence of SV40 DNA. Nature (London) 273, 113-120. GOODING, L. R. (1977). Specificities of killing by cytotoxic lymphocytes generated in viva and in vitro to syngeneic SV40-transformed cells. J. Immund 118, 920-927. HABEL, K., and EDDY, B. E. (1963). Specificity of resistance to tumor challenge of polyoma and SV40 virus-immune hamsters. Proc. SW. Ezp. BioL &fed 113, 1-4. HENNING,
R., LANGE-MUTSCHLER,
J., and
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