Expression of tumor-specific surface antigens on cells infected with temperature-sensitive mutants of avian sarcoma virus

VIROLOGY

64,400-408

Expression

(1975)

of Tumor-Specific

Surface

with Temperature-Sensitive REINHARD

’ The imperial

KURTH,

Mutants

1 ROBERT HEINZ

Antigens

on Cells Infected

of Avian Sarcoma

R. FRIIS, 2 JOHN BAUER2

A. WYKE, l

Cancer Research Fund Laboratories, London WC2A 3PX, England, and 1Institut Fachbereich Humanmedizin, Justus Liebig Universitiit, 63 Giessen, W. Germany Accepted

November

Virus AND

ltir Virologie,

4, 1974

Tumor-specific surface antigens (TSSA) have been found in all avian sarcoma virustransformed cells so far examined. We have studied the expression of these antigens in cells infected with mutant viruses and in which a phenotypically normal or transformed state depends on the temperature of incubation. We found that in cells infected with these mutants, all of which are defective in the ability to induce host cell transformation at the nonpermissive temperature, there are differences in the expression of TSSA. The questions whether TSSA are viral-coded functions and whether they are directly involved in the maintenance of the transformed phenotype are discussed in the light of our results. INTRODUCTION

Malignant cell transformation by RNA tumor viruses leads to the insertion of new molecules into the cell plasma membrane possibly accompanied by modification of existing membrane components. Upon transplantation of transformed cells into syngeneic hosts at least some of the new or altered molecules are sufficiently immunogenic to induce a detectable immune response. Such molecules can be divided into viral structural components, which are situated at virus budding sites and throughout the cell membrane, and new cell membrane components which are apparently not incorporated into the virion envelopes. Some of the virus-induced membrane components appear to be strictly tumor cell spedific and because of their immunogenicity are called tumor-specific cell surface antigens (TSSA; for a recent review see Bauer, 1974). In the avian RNA tumor virus (ATV) system TSSA have been demonstrated in the cell membranes of all transformed cell cultures, regardless of the ATV strain used for transformation and of the species of the 400

Copyright 0

1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

transformed cells (Kurth and Bauer, 1972a,b; Gelderblom et al., 1972, Gelderblom and Bauer, 1973). It is not known whether the transforming virus codes directly for these molecules or whether they are virus-induced cell gene products, e.g., tumor antigens of embryonic origin (Kurth and Bauer, 1973a). They may also represent a combination of virus and cell-coded products. The function, if any, of the tumor-specific cell surface components is an even more relevant question, for these molecules might share responsibility for the release of tumor cells from growth control. The use of transformation-defective virus mutants may help in elucidating the questions of origin and function of TSSA. Temperature-sensitive (ts) ATV mutants which are defective in their transformation and/or replication functions have recently been isolated and characterized (reviewed in Tooze, 1973; Wyke, 1974). Our study describes the temperature sensitivity of TSSA and other transformation markers in cells infected by a number of these mutants.

TUMOR-SPECIFIC MATERIALS

AND

SURFACE

METHODS

Cells. Chicken embryo cells (CEC) were prepared from 11-day-old specific pathogen-free chicken embryos generously supplied by Dr. E. Vielitz, Lohmann Tierzucht, Cuxhaven, Germany. CEC were infected with cloned stocks of mutants and were transferred at least twice so that confluent infection was obtained. Cultures of transformation-defective mutants were routinely incubated at 41”, the nonpermissive temperature, so that cell morphology was normal. Coordinate mutants were maintained during the initial infection phase at the permissive temperature (35”) to establish confluent virus infection; thereafter cultures were propagated at 41”. Experiments were performed using different techniques to quantitate the state of transformation. Focus tests were performed as described by Vogt (1969) according to the method of Rubin (1960), soft agar suspension cultures were prepared as previously reported (Friis, 1972), and assays for the rate of uptake of radioactively labeled 2-deoxyglucose were done as described by Graf and Friis (1973). For use in the direct or indirect antibody-labeling techniques, normal and infected CEC derived from a single embryo were seeded into the 96 (16-mm) wells of Linbro plates No. 96-CV-TC (Linbro Chemical Co. New Haven, Conn.) Cells were maintained for 10 hr at the nonpermissive temperature (41”) to allow them to attach, then half of the cultures were shifted down to 35” for 18 hr to allow expression of transformation by the mutant-infected CEC. Cells were then washed once in PBS and fixed with 0.125% glutaraldehyde for the tests. Those wells destined to remain at 41” were seeded with 8 x 10’ CEC while those to be shifted to 35” were seeded at 12 x 10’ per well. This procedure resulted in approximately equal cell numbers of between 2.5 and 3.0 x 105/well at the time the CEC were fixed. For cell washes, the entire plate was immersed in PBS, transferred for 5 min to a gentle rocking platform after which PBS was poured off by inverting the plate.

ANTIGENS

401

Viruses The isolation and physiologic properties of the transformation-defective temperature-sensitive mutants of Prague strain Rous sarcoma virus (RSV) have been described (Wyke, 1973a; Wyke and Linial, 1973). These mutants, ts LA22 to ts LA34 replicate normally at nonpermissive temperature but cannot transform cells under these conditions. They have been divided into four cooperative-transformation groups (Wyke et al., 1974; see Table 2). Ts LA336m a mutant of B77 strain avian sarcoma virus, was isolated by Toyoshima and Vogt (1969); and it has been shown to be a coordinate mutant defective in an early function (Friis et al., 1971), probably in the reverse transcriptase-ribonuclease H activity (MSlling and Friis, in preparation). There is evidence to suggest that ts 336m may have a second temperature-sensitive lesion affecting a late transformation function (Friis et al., 1971; Wyke and Linial, 1973; Friis, unpublished observations), the suffix m denoting a virus with known or suspected multiple mutations. Ts LA336m is the only one of the coordinate mutants used here which produces viruses at 41’ after initiation of infection at 35 “. Ts LA334m was originally obtained by Toyoshima and Vogt (1969) from B77 and has been characterized as a mutant defective in late replication and transformation functions (Friis et al., 1971). Ts 334m has been recently shown to have two ts lesions, one affecting only replication of the virus and the other transformation (Owada and Toyoshima, 1973). Ts LA339 is a coordinate ts mutant of B77; its properties have been reported in Graf and Friis (1973). The wild-type parental viruses used for control purposes were: Prague strain rous sarcoma virus subgroup A (PR-A) and avian sarcoma virus B77, subgroup C. Sera. Anti-TSSA sera were prepared as previously described (Kurth and Bauer, 1972a) by injecting subtumorigenic doses of wild-type Schmidt-Ruppin virus H (SRV-H; subgroup D) into the wing web of adult L-15 chickens. This virus was originally isolated from a hamster tumor in-

402

KURTH ETAL.

duced by RSV (Bauer and Graf, 1969). It has been demonstrated that immunized birds develop antibodies both against virus structural components, i.e., mainly against viral envelope antigens (Ve-Ag), as well as against virus- and tumor-associated embryonic antigens (Kurth and Bauer, 1973a). Since all tested CEC were infected with either wild-type Prague strain A (subgroup A), wild-type B77 (subgroup C), or mutants derived therefrom (all in subgroups A and C), the above antiserum will not react with the serologically distinct subgroups A and C envelope antigens expressed on the surface of infected CEC target cells (Gelderblom et al., 1972). Those sera with the highest virus-neutralizing titer generally also possessthe highest titer of TSSA-specific antibodies. Normal chicken serum from birds of the same randomly inbred L-15 flock served as negative control. For the indirect antiglobulin technique, rabbits were immunized with a total of 1.5 mg isolated chicken immunoglobulin emulsified 1:l in Freund’s complete adjuvant and injected subcutaneously at different sites. The rabbits were first bled 4 wk after immunization. Booster injections were given intravenously. Isolation lins

and Iodination

of Immunoglobu-

For both the direct and indirect labeledantibody technique, chicken or rabbit antibodies were purified by precipitation with 35% final concentration of ammonium sulfate followed by G-200 chromatography. After concentration of the second peak of the eluate and Lowry determination of the protein content, approximately 1 mg IgG in 20 ~1 PBS was iodinated with 1 mCi of either [Y]Na or [lslI]Na (carrier-free) by the addition of 5 ~1 PBS containing 60 pg chloramine T. After 2 min the reaction was stopped by adding 5 ~1 (250 1.18)sodium metabisulfite and the mixture was immediately chromatographed on a small G-25 column to separate labeled IgG from unbound reactants. The specific activity amounted usually to approximately 0.8 pCi/pg protein. The IgG preparations were

always absorbed (60 minl4” with gentle rocking) to monolayers of chf-negative chicken fibroblasts fixed with 0.125% glutaraldehyde and centrifuged (200,000 g/45 min) thereafter to remove antigen-antibody complexes and debris. The IgG content of the sample was again determined by measuring its radioactivity to correct for loss during absorption. PRILAT. It is the inherent advantage of the paired radioiodine-labeled antibody technique (PRILAT) that target cells are incubated with both normal and specific immunoglobulins at the same time. For that purpose normal and immune antibodies are labeled either with ‘31iodide or ‘Yodide which can be distinguished in /3or y-scintillation counters. Since it is a well-established observation that highly iodinated proteins tend to absorb nonspecifically to other proteins, care was taken that equal amounts of IgG were iodinated under identical conditions with equal nanogram amounts of carrier-free [“‘I]Na or [1311]Na (Radiochemical Center, Amersham U.K.). Equal quantities of normal and specific IgG were then mixed to give the paired label mixture (PLM). The portion of nonspecific binding mimicking specific absorption of immunoglobulins to appropriate target cells can then be monitored directly by incubating the fixed target cells in the 16-mm Linbro wells with increasing dilutions of 0.1 ml PLM (30 min/20” on a gentle rocking platform). Incubation was terminated by aspirating the PLM and washing the wells by immersing them four times in PBS, each for 5 min. The final wash consisted of 0.4 ml KCl/ HCl buffer (pH 1.0) for 5 min to break up antigen-antibody interaction (Sjijgren et al., 1971). Three-tenths milliliter of the buffer was transferred to scintillation vials and counted for both lz51and 1311content. Target cell number per well was determined from parallel wells to allow calculation of antibody binding per cell knowing the specific radioactivity of the IgG preparation and assuming a molecular weight of 150,OOOdfor the IgG. Specific absorption was defined by the difference of binding of specific IgG minus unspecific binding of normal IgG.

TUMOR-SPECIFIC

SURFACE

PRILAT has the disadvantage that isolation and especially iodination of immunoglobulins inevitably results in some loss of titer. Therefore, we also used the indirect anti-globulin technique, incubating parallel target cell cultures first with either normal or anti-TSSA chicken serum and then with iodine-labeled rabbit antichicken IgG immunoglobulins. Since the indirect technique essentially confirmed the results obtained before with the PRILAT, only the PRILAT results will be illustrated below. RESULTS

Various parameters of transformation were examined with different mutants to determine which assays for the transformed state were the most invariable indicators of temperature sensitivity. Table 1 shows that the ability to form foci in monolayers of CEC represents the most stringent measure of the temperature sensitivity of the transformed state. This is perhaps not unexpected since these mutants were selected on the basis of changes in this property. Interestingly, the formation of colonies in soft agar suspension cultures and growth to a high cell density do not always parallel this inability to form foci, findings already noted for ts 334m and TABLE

403

ANTIGENS

for ts 25 (Friis et al., 1971; Wyke and Linial, 1973). The rate of uptake of certain sugars, e.g., 2-deoxyglucose, is also a measure of transformation which correlates closely with focus formation, with the possible exception of ts 25. These assays for transformation were applied to cultures prepared for testing for the presence of TSSA. In the direct antibody-labeling technique (PRILAT) uninfected control as well as fully infected CEC were tested with a PLM consisting of equal amounts of normal and anti-SRV-H chicken IgG. The same IgG preparations were used throughout all experiments. In pilot experiments it had previously been demonstrated that the anti-SRV-H chicken serum detects TSSA on wild-type Prague A virus-transformed CEC. Figure 1 shows the specific absorption of anti-TSSA antibodies to mutant infected cells at 35”. At this permissive temperature when mutant infected cells are transformed the quantitative absorption of antiTSSA antibodies was practically indistinguishable from the absorption to wild-type transformed cells. Thus, the induction of TSSA at 35” coincides with the expression of the other parameters described above (Table 1) that are characteristic for the transformed state of the cells. The max1

PARAMETERSOF TRANSFORMATIONUSED TO ASSESSTHE TEMPERATURE SENSITIVITY OF MUTANT INFECTED CELLS Virus

None PR-A wt Bllwt ts 23 ts 24 ts 25 ts 29 ts 33 ts 334m ts 336m ts 339

Focus formation FFU/ml41” FFWml35” 1.20 0.70 0.0003 0.005 0.002 0.0002 0.0001 0.004 0.0009 0.012

Colony formation CFU/ml41” CFU/ml35” 0.80 0.50 0.010 0.001 0.70 0.090 0.008 0.42 0.016 0.008

Counts per min [2-3H]deoxy glucose uptake per lo6 cells 35”

41°

6,000 51,000 34,000 30,000 39,000 48,000 26,000 33,000

g,oocJ 63,000 41,000 8,000 9,000 16,000 5,000 8,000 ND ND ND

Relative cell density at nonpermissive temperature” 1 1.9 1.3 0.9 1.1 2.1 1.0 1.6 ND ND ND

a Cell density was estimated at 72 hr after inoculation of plates at an initial density of 4 x lo5 cellsAO-mm plate. Data are expressed as the ratio of cell number of mutant infected cells to the number of normal cells in cultures maintained at 41’. PR-A wild-type and B77 wild-type infected cultures show an artificially low cell density because of loss of rounded transformed cells into the medium.

404

KURTH

‘I 4 d

-TV----55 1, 22 44 PLlv C311ieltr0’10” ,pg ‘01 rr )

2 75

FIG. 1. PRILAT. Absorption of radioactivity labeled immunoglobulins from normal and anti-SRV-H chicken sera to chicken ‘embryo cells infected by temperature-sensitive avian oncornavirus mutants. Target cells are grown at the permissive temperature (35”). x-x: absorption to uninfected, chf-negative CEC. O-----O: absorption to CEC infected by wild-type Prague A- or B77-viruses or by the tsmutants 23, 24, 25, 29, 31, 33, 334m, 336m, 339 derived therefrom. Specific absorption = binding of immunoglobulins from SRV-H-immunized chickens minus binding of immunoglobulins from unimmunized chickens. The standard deviation indicates the range of the absorption of the CEC infected with the various mutants. Ordinate: number of immunoglobulins absorbed per cell. Abscissa: total concentration of immunoglobulins in PLM.

imum absorption of anti-TSSA antibodies to transformed CEC ranges between 2.e and 3.6 x lo4 IgG molecules per cell. Probably twice as many TSSA-receptors are, in fact, inserted into the cell membrane, since evidence exists that the IgG are bound divalently to the cell surface because they induce patch formation of TSSA which can be visualized in fluorescent pictures (Kurth and Bauer 197213,and unpublished observations on stained chicken cells). These quantitative data represent minimum estimates for two reasons. First, the target cells grow on substrate and probably the antibodies cannot reach the cell surface area attaching to the plastic dish after fixation. Second, the incubation of cells with PLM requires four final washes to eliminate nonspecific antibody binding. This procedure is bound to remove also some specifically absorbed anti-TSSA immunoglobulins, especially those with low avidity.

ET AL.

Figure 2 represents the absorption pattern of mutant infected cells at the nonpermissive temperature of 41”, when cells exhibit a normal phenotype (Table 1). Not all mutant infected CEC showed a loss of TSSA expression at the nonpermissive temperature, despite their normal cell morphology. The notable exceptions are the mutants ts 23, ts 24, and ts 31, which at both 35” and 41” induce quantitatively as many TSSA receptors as the wild-type viruses. The ts 25 mutant is a special case since, as reported by Wyke and Linial (1973) and shown above (Table l), it expresses the temperature-sensitive function to some degree also at 41”. It is, therefore, not surprising to find a low TSSA-induction at the nonpermissive temperature in the case of this mutant. CEC infected by the other two T-class mutants, ts 29 and ts 33 show a residual absorption of very few c ‘g

, z?-

FIG. 2. PRILAT. Text as in Fig. 1, except that target CEC were cultured at the nonpermissive temperature (41”). x-x: absorption to uninfected CEC or CEC infected by the ts-mutants 334m, 336m, 339. A. .A: absorption to CEC infected by the ts-mutants 29, 33. O-.-,-Cl: absorption to CEC infected by the ts-mutant 25. O-----O: absorption to CEC infected by wild-types Prague A- or B77-strains or by the ts-mutants 23, 24, 31. Specific absorption = binding of immunoglobulins from SRV-H-immunized chickens minus binding of immunoglobulins from unimmunized chickens. The standard deviation indicates the range of the absorption of the CEC infected with the various mutants. The titration data are taken from a single experiment which is representative for a series of similar determinations. Ordinate: number of immunoglobulins absorbed per cell. Abscissa: total concentration of immunoglobulins in PLM.

TUMOR-SPECIFIC

SURFACE

ANTIGENS

405

tive in that they behave like the wild-type parental strains at 35”, whereas at 41’ they are unable to transform CEC. The transforming abilities of the tested ts-mutants were demonstrable by comparing morphology, focus, and colony formation as well as uptake of [2-3H]deoxyglucose of mutant and wild-type infected cells at 35” and 41” (Table 1). Parallel cultures of mutant and DISCUSSION wild-type infected CEC kept at either 35” The induction of tumor-specific surface or 41” were then tested for expression of antigens by transformation-defective avian TSSA. At the permissive temperature of 35 ‘, all oncornavirus mutants has been investigated. The mutants are temperature sensi- tested ts-mutants induced as many TSSAreceptors as the corresponding wild type. There was no consistent quantitative difTABLE 2 ference in TSSA induction detectable at TSSA EXPRESSION IN CHICKEN EMBRYO FIBROBLASTS 35 ‘. Thus, TSSA expression corresponded INFECTED BY TEMPERATURE-SENSITIVE MUTANTS OF fully to the transformed phenotype of all AVIAN SARCOMA VIRUSES wild-type and mutant infected cells. UninTemperTSSA expression Mutant COfected CEC, serving as negative control, aturenumber operative sensitive 35” Per- 41” Nontransusually adsorbed a low amount of antiphenomissive permissive formation bodies from the anti-TSSA serum (Fig, 1). type” WUP” The reason for this background absorption is not known, especially since both normal Uninfected and antisera were preabsorbed with the Wild-type ++ ++ Prague A uninfected control CEC to rule out an Wild-type ++ ++ absorption due to the influence of naturally B77 occurring alloantibodies or calf serum conI ts 25 T ++ + taminants in the SRV-H virus preparation I * ts 33 T ++ used for antiserum production. T II ++ ts 23 +t At the restrictive temperature of 4l”, ts 24 III T ++ ++ mutant infected CEC with the exception of III T ts31 ++ ++ the ones infected by the truly leaky ts 25 IV T * ts 29 ++ ts 334m’ C and ts 334m mutants were phenotypically ++ ts 336m’ C ++ as normal as the uninfected CEC (Table ts 339 C ++ 1). The results of TSSA expression, however, were more complex (Table 2). Again, ” Cooperative transformation groups are defined by wild-type infected and uninfected CEC the ability of T-class mutants to induce cell transforserved as positive and negative controls, mation upon double infection at nonpermissive temrespectively. The mutants can be classified perature under conditions where subsequent reinfection is permitted. Double infections by mutants in into several groups according to their abildifferent groups show this effect, while double infecity to induce TSSA at restrictive temperations by mutants within a group do not do so (Wyke. ture. The three tested C-class mutants (ts 1973b). 334m, ts 336m, ts 339) are unable to induce b T = T-class mutants, transformation-defective, TSSA under nonpermissive conditions. In replication unaltered; C = C-class mutants, transforthese casesthe lack of TSSA induction is in mation- and replication-defective. full accordance with the normalized pheno<‘The suffix m denotes mutants with known or type of the mutant infected CEC. suspected multiple mutations whose coordinately deIn most of the experiments, two T-class fective phenotype may thus represent a complex of mutants, ts 29 and ts 33, induced a slight replication-defective, transformation-defective, and coordinately-defective lesions. absorption of immunoglobulins from the immunoglobulin molecules under nonpermissive conditions. All three coordinate mutants tested, i.e., ts 334m, ts 336m, and ts 339 show no TSSA induction under nonpermissive conditions. The results of the TSSA inducibility by the different mutants are summarized in Table 2.

406

KURTH ETAL.

anti-TSSA serum, the cause of which is not clear. It must, however, be kept in mind that virus replication is unaltered in Tclass mutant infected CEC. If, as in the murine oncornavirus system (Ferrer, 1973; Yoshiki et al., 1973), virus replication leads to the insertion of group-specific virus structural proteins into the cell membrane, one would expect that anti-SRV-H antibodies in the anti-TSSA serum will bind to these proteins. ts 25 is a special case in that it shows residual transforming functions even at 41’: more refractile morphology, a slightly increased sugar uptake compared to uninfected CEC, the ability to grow in suspension agar, and to grow to a high cell density on a solid substrate. Here it is now shown that ts 25 also induces a low level of TSSA at the restrictive temperature. It came as a surprise to find that ts 24 and ts 31 (both in cooperative transformation group III) and ts 23 (cooperative transformation group II) are able to induce TSSA under nonpermissive conditions in otherwise phenotypically normal cells. Two interpretations are possible. First, it is conceivable that TSSA molecules themselves are the ts-gene product of the mutant and at 41” could still be immunologically intact by functionally defective. This would allow their detection by immunological methods while their yet unknown function is impaired. This hypothesis is particularly attractive if TSSA represent tumor cell surface-associated enzymes, e.g., proteases or glycosyl-transferases, such as have been described by several laboratories to be present on transformed cells (reviewed in Tooze 1973; Hynes 1974). Second, all three TSSA-positive mutants are late mutants and virus replication at 41” remains unchanged. One can, therefore, expect that some (e.g., TSSA), but not all transforming proteins are synthesized at that temperature and in the case of the TSSA-positive mutants the ts-defect preventing the successful completion of the transformation process may occur at a stage later than TSSA induction. These tumor antigens, therefore, may

be a parameter or even a prerequisite for cell transformation, but if this second hypothesis is correct, they themselves are not sufficient to establish and maintain the phenotype of tumor cells. These findings represent the second instance in which tumor antigens could be detected on phenotypically normal cells. It had been shown previously that increasing the intracellular level of cyclic AMP in tumor cells leads to normalization of tumor-associated parameters, i.e., restoring normal morphology, agglutinability by lectins, and possibly growth control. TSSA expression, however, remained positive (Kurth and Bauer 1973b). One could, therefore, speculatively, rephrase the definition of TSSA in that they are not markers for the transformed state of a cell, but for the intracellular presence of a transforming agent whose other functions may be suppressed under appropriate experimental conditions (Kurth and Bauer, 1974). The number of ts-mutants investigated is still too small to allow a decision on whether the ability to induce TSSA can be used as a viral genetic marker. It may still be accidental that mutants of the two cooperative transformation groups II and III are positive for TSSA at 41” whereas those of groups I and IV are negative in that connection. The failure to differentiate the four cooperative transformation groups by other physiological criteria, and the possibility that cooperative transformation results from genetic recombination rather than complementation (Wyke et al., 1974) suggests that the T-class mutants may all be defective in the same function. The behavior of TSSA is the only parameter which so far distinguishes the groups, but only after additional mutants have been checked can it be decided whether TSSA induction can be used as a marker for mapping the viral genome. To our knowledge, this is the first report in any tumor virus system to describe the effect of transformation-defective tsmutants on tumor cell surface antigens. The significance of the finding of TSSApositive mutants lies in their potential use

TUMOR-SPECIFIC

as vaccine viruses: they are unable to transform but able to induce tumor antigens. It is obvious that temperature-sensitive mutants of oncogenic viruses which are subject to frequent reversion are too dangerous to be used for vaccination purposes, but the now established fact of their existence is a great encouragement to start looking for TSSA-positive deletion mutants that are defective in transformation. Such mutants would need to be strictly nononcogenic and unable to undergo complementation or recombination with endogenous viruses. Thus, they may be of value in establishing methods to prevent immunological enhancement and to induce cytotoxic activity against tumors. ACKNOWLEDGMENTS The authors thank Mrs. I. Wolf and Mrs. M. L. Bergerhausen for excellent technical assistance. We also express our appreciation to Dr. I. Macpherson for advice and valuable discussions and for critically reading the manuscript. REFERENCES BAUE~ H., and GRAF, T. (1969). Evidence for the existence of two envelope antigenic determinants and corresponding cell receptors for avian tumor viruses. Virology 37,157-161. BAUER, H. (1974). Virion and tumour cell antigens of C-type RNA tumour viruses. Aduan. Cancer Res. 20, in press. FERRER, J. F. (1973). Cell-surface and virion-envelope antigen shared by radiation leukemia virus (Rad LV) and other murine C-type viruses. Znt. J. Cancer 12, 378-388. FRIIS, R. R., TOYOSHIMA, K., and VOGT, P. K. (1971). Conditional lethal mutants of avian sarcoma viruses. I. Physiology of ts 75 and ts 149. Virology 43, 375-389. FRIIS, R. R. (1972). Abortive infection of Japanese quail cells with avian sarcoma viruses. Virology 50, 701-712. GELDERBLOM,H., BAUER, H., and GRAF, T. (1972). Cell surface antigens induced by avian RNA tumor viruses: Detection by immunoferritin techniques. Virology 47, 416-425. GELDERBLOM, H., and BARER, H. (1973). Common avian oncornavirus-induced tumor antigens in different species as revealed by immunoferritin-techniques. Znt. J. Cancer 11, 466-472. GRAF, T., and FRIIS, R. R. (1973). Differential expression of transformation in rat and chicken cells infected with an avian sarcoma virus ts mutant.

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Virology 56, 369-374. HYNES, R. 0. (1974). Role of surface alterations in cell transformation: the importance of proteases and surface proteins. Cell 1, 147-156. KURTH, R., and BAUER, H. (1972a). Cell surface antigens induced by avian RNA tumor viruses: Detection by a cytotoxic microassay. Virology 47, 426-433. KURTH, R., and BAUER, H. (1972b). Common tumorspecific surface antigens on cells of different species transformed by avian RNA tumor viruses. Virology 49, 145-159. KURTH, R., and BAUER, H. (1973a). Avian oncornavirus-induced tumor antigens of embryonic and unknown origin. Virology 56, 496-504. KURTH, R., and BAUER, H. (1973b). Influence of dibutyryl cyclic AMP and theophylline on cell surface antigens of oncomavirus transformed cells. Nature New Biol. 243, 243-245. KURTH, R., and BAUER, H. (1974). Avian RNA tumor viruses: A model for studying tumor associated cell surface alterations. BBA Reviews on Cancer, in press. OWADA, M., and TOYOSHIMA, K. (1973). Analysis on reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B 77. Virology 54, 170-178. RUBIN, H. (1960). A virus in chick embryos which induces resistance in uitro to infection with Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 46, 1105-1119. SJBCREN, H. O., HELLSTRBM, I., BANSAL, S. C., and HELLSTRBM, K. E. (1971). Suggestive evidence that the “blocking antibodies” of tumor bearing individuals may be antigen-antibody complexes. Proc. Nat. Acad. Sci. USA 68, 1372-1375. TOOZE, J. (ed. 1973). “The Molecular Biology of Tumour Viruses.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. TOYOSHIMA, K., and VOGT, P. K. (1969). Temperature sensitive mutants of an avian sarcoma virus. Virology 39,930-931. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York. WYKE, J. A. (1973a). The selective isolation of temperature-sensitive mutants of Rous sarcoma virus. Virology 52, 587-590. WYKE, J. A. (197313). Complementation of transforming functions by temperature-sensitive mutants of avian sarcoma virus. Virology 54, 28-36. WYKE, J. A., and LMIAL, M. (1973). Temperature sensitive avian sarcoma viruses: a physiological comparison of twenty mutants. Virology 53, 152-161. WYKE, J. A. (1974). The genetics of C-type RNA

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tumor viruses. Znt. Reu. Cytol. 38, 67-109. J. A. (1974). Genetic recombination among temperature sensitive mutants of Rous sarcoma virus. Cold Spring Harbor Symp. Quant. Biol. 39, in press.

WYKE, J. A., BELL, J. G., and BEAMAND,

ET AL. T., MELLOW, R. C., and HARDY, W. D. (1973). Common cell-surface antigen associated with murine and feline C-type RNA leukemia viruses. Proc. Nat. Ad. Sci. USA 70, 1878-1882.

YOSHIKI,