Int. J . Cancer: 21, 490-495 (1978)
MAMMARY LEUKAEMlA (ML) ANTIGEN ISOLATED FROM L 1210 LEUKAEMIA CELLS Teresa ZAK-NEJMARK, Jadwiga STEUDEN and Czeslaw RADZIKOWSKI Department of Tumour Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
Crude 3 M KCI extracts from L 1210 cells and purified M L antigen were characterized and the presence of M L antigens was verified by various antiM L sera, as well as by antisera to disrupted M M T V B particles. Selected ML-positive fractions from preparative polyacrylamide electrophoresis and purified M L antigen isolated by affinity chromatography were analyzed by polyacrylamide disc electrophoresis. W i t h both procedures the same sharp band was obtained. The molecular weight of both preparations was found t o be 73,500 as estimated. The relationship between M L antigen, M M T V antigenic products and cell-associated viral proteins i s discussed.
The mammary leukaemia (ML) antigen is the mouse mammary tumour virus (MMTV)-related cellular membrane antigen characteristic of cells of leukaemias and mammary cancers induced or occurring naturally in DBAJ2, C3H, A and GR mice. This antigen was discovered by Stuck et al. in a direct complement-dependent cytotoxic test with the use of alloantisera produced in C57BL/6 mice by immunization with DBA/2 leukaemia cells (Stuck et al., 1964). In our previous paper (Steuden et al., 1978) we demonstrated the presence of ML antigen on leukaemia L 1210 cells by using both heterologous (rabbit) and alloantisera (BALB/c) and anti-ML sera produced in syngeneic DBA/2 mice. In addition, by applying appropriate absorptions and blocking techniques combined with ferritin-labelled antibodies in electron microscopy studies (EM), we demonstrated the independent localization of ML antigen and murine leukaemia virus-Gross (MuLV-G) cellular antigen on L 1210 cells (Steuden et al., 1978). The ferritin-labelled antigenic sites appeared to be free from budding viral particles. It should be mentioned that in our EM studies no MMTV B particles but abundant intracytoplasmic A particles (B particle precursors) and MuLV C particles could be demonstrated (Radzikowski et al., 1972). An interrelationship and/or identity between ML and MMTV antigenic products is emphasized by several authors. Positive serological reactions with ML-positive cells were observed not only with antiML sera but also with antisera obtained by immunization of rabbits with isolated, ether-disrupted and purified B particles (Nowinski et al., 1967, Hilgers et al., 1975; Van Blitterswijk et al., 1975; Sarkar et a/., 1971). There is, however, no convincing evidence available that antisera obtained in syngeneic, allogeneic and xenogeneic hosts identify the same antigen or antigenic complex on leukaemic cells.
Thus, it seems appropriate, as suggested by Hilgers et al. (1975), to use the name ‘‘ MLm ” for the antigen identified by antisera produced in mice and “ MLr ” for the antigen identified by heterologous (rabbit) antisera against MMTV B particles. In the present studies the 3~ KCI extracts of leukaemia cells purified by polyacrylamide gel electrophoresis (PAGE) and the antigen from leukaemia L 1210 cells purified by affinity chromatography were characterized and their identity verified by anti-ML sera, obtained from both allogeneic and xenogeneic hosts. Heterologous antisera against disrupted MMTV B particles were also used (Steuden et al., 1978). The use of these latter antisera seems especially valuable, if one considers that the DBA/2 leukaemia L 1210, contrary to the GR leukaemia GRSL/2 (Hilgers et al., 1973, 1975) was found to produce no budding MMTV B particles. The positive serological membrane reactions of L 1210 cells with anti-MMTV disrupted B particle antisera could occur only if MMTV antigenic products were present on the cell surface. MATERIAL A N D METHODS
Animals Mice of all strains used were maintained by brother xsister mating at the Inbred Mice Center of the Institute of Immunology and Experimental Therapy in Wroclaw. Their main characteristics were: DBA/2 (H-2d) and C3H (H-2k) with high natural incidence of mammary tumours. BALB/c (H-2d), C57BL/6 (H-2b) with low natural tumour incidence, AKR (H-2k) with high natural incidence of leukaemia. Rabbits were obtained from the breeding colony maintained at the same Institute. Tumours L 1210 V. This transplantable DBA/2 leukaemia has been maintained at the Department of Tumour Immunology since 1969 as an ascites and a solid transplantable line. The presence of ML antigen on leukaemia cells and production of a large number of intracytoplasmic viral A particles and MuLV C particles are constant traits of these cells. We have never detected any MMTV B particles, either budding or extracellular. The cells from either ascites or leukaemic spleens were harvested, as a rule, 4 days after inoculation of 5-10 x lo6 cells intraperitoneally, or sometimes, subcutaneously. Leukaemia L 1210 cells were used for preparation of 3 M KCI extracts and purified antigenic material.
Received: December 21, 1977.
CHARACTERIZATION OF ML ANTIGEN
E 0" G2. This transplantable C57BL/6 lymphoma, maintained at our Department as a transplantable line since 1976, was received from Dr. E. A. Boyse, Sloan-Kettering Cancer Institute, New York, N. Y. The presence of Gross cellular surface antigen (GCSA) as well as of other MuLV proteins, is a constant feature of the leukaemia cells. This leukaemia line and cells from naturally occurring AKR leukaemias were used as GCSA-positive reference lines for absorption of anti-MuLV-Gross antibodies from oligovalent antisera produced by immunization with leukaemia L 1210 cells. Cells from naturally occuring mammary tumours in C3H and DBA/2 mice were used as ML-positive reference lines for absorption or as controls in cytotoxic tests. Antisera Alloantisera against ML antigen (anti-ML sera) were prepared by immunization of BALB/c (H-2d) mice with leukaemia L 1210 (H-2d) cells. These sera reacted in cytotoxicity tests with ML-positive cells of spontaneous mammary tumours of DBA/2 and C3H mice and L 1210 leukaemia, but were not toxic to normal DBA/2 lymphoid cells. Cytotoxic anti-ML antibodies could be removed by absorption with MLpositive cells of spontaneous or transplantable mammary tumours and/or of leukaemia L 1210. The anti-ML sera did not react in cytotoxicity tests with leukaemia cells of Gross-positive spontaneous AKR lymphomas or transplantable E 0' G2 leukaemia in C57BL/6 mice. The anti-ML cytotoxic activity could not be removed by absorption with Gross-positive leukaemia cells. Immunization schedules and serological characterization of these sera are described elsewhere (Steuden et al., 1977). Anti-H-2d serum (C57BL/6 anti-BALB/c) was kindly provided by Dr. A. Lengerova of the Institute of Molecular Genetics, Prague. Heterologous anti-ML sera were produced in rabbits by immunization with leukaemia L 1210 cells as described elsewhere (Radzikowski et al., 1977).After repeated absorption with normal DBA/2 cells, these sera reacted in cytotoxic tests exclusively with ML-positive cells. Normal DBA/2 lymphoid cells and Gross-positive AKR and Eo" G2 leukaemia czlls were used as negative controls. Soluble antigen preparation The preparation of 3 M KCI extracts was a modification of the method used by Reisfeld et al. (1971) for solubilization of transplantation antigens. Leukaemia L 1210 cells were washed twice with cold phosphate-buffered saline (PBS, PH 7.4). Washed cells were resuspended in cold 3 M KCI in PBS at a concentration of los cells in 20 ml and gently agitated for 16 h at 4" C. After centrifugation at 165,OOOg for 1 h, the supernatant was separated from the viscous pellet and dialyzed against 200-fold volumes of cold PBS for 48 h with three changes. The dialyzate was clarified by centrifugation at 150,000g for 20 min and concentrated by ultrafiltration. Crude soluble antigen preparations were stored at -20" C and used for preparative polyacrylamide gel electrophoresis and affinity chromatography.
49 1
Preparative polyacrylamide gel electrophoresis Preparative, discontinuous polyacrylamide gel electrophoresis was used to purify the antigenic principle. Electrophoresis was performed in buffer system B of Rodbard and Chrambach (1971) at 4" C at constant current 20mA. A sample of 50mg protein was applied to the gel. After elution with PBS, fractions of 1.7 ml each were collected when the tracking dye (0.05 % Coomassie brilliant blue) had reached the end of the gel. Protein was determined in all fractions and total carbohydrate, aminosugar and sialic acid were determined in selected fractions. Absorption capacity of all fractions was tested in cytotoxic neutralization assay. Polyacrylamide gel disc electrophoresis The samples of crude soluble antigen were mixed with urea, SDS and mercaptoethanol at a final concentration of 8 M, 0.1 % and 2 % respectively. The mixture was heated to 100" C for 2 min and applied directly to the cooled gel. Samples were electrophoresed in the presence of SDS as described by Weber and Osborn (1969). Gels were stained with Coomassie brilliant blue and periodic acid-Schiff (PAS). Molecular weight of the ML antigen was estimated from its relative migration in gels by coelectrophoresis with standard molecular weight markers: cytochrome C (12,400), horse myoglobin (1 7,8OO), bovine serum albumin (67,000) (Serva, Heidelberg, West Germany), trypsin (23,300), ovalbumin (43,000) (Sigma, St. Louis, USA) and galactosidase (1 30,000) (Biochemicals, Wisconsin, USA). Molecular weights were calculated according to Weber and Osborn (1969) by the following formula: Distance of Drotein Length - before migration staining Mobility = X Length after Distance of dye destaining migration The mobilities were plotted against the known molecular weight expressed on a semi-logarithmic scale. Afinity chromatography Immunoabsorbent was prepared by the technique of Jansen etal. (1975). A column (K9/15 Pharmacia, Uppsala, Sweden) of CNBr-activated Sepharose 4B (Pharmacia) coupled with rabbit anti-ML globulins was used. The solution containing the ML antigen (crude 3 M KCI extracts from L 1210 leukaemia cells) was applied to the column at the rate of 20 ml/h. Absorbed antigen was released by , 7.6) containing 2 M Tris-HCI buffer (50 m ~ PH MgClz and immediately afterwards dialyzed against PBS for 24 h. The dialyzed eluate was concentrated by vacuum ultrafiltration up to 0.1 mg protein per 1 ml. Protein estimation The method of Lowry et al. (1951) was used with bovine serum albumin as a standard or the total concentration of protein in the cell suspension was determined according to the method of Oyama and Eagle (1956).
492
ZAK-NEJMARK ET AL.
Neutralization assay for soluble antigen absorption capacity The neutralization of anti-ML cytotoxic activity from allo- and heterosera was performed as follows : serial dilutions of anti-ML sera were incubated with successive electrophoretic fractions of purified antigenic preparations and in a parallel experiment with various numbers of L 1210 cells for 1 h at 37" C. Then complement-dependent cytotoxic tests according to the method of Gorer and O'Gorman (1956) were performed. Sugar composition Sugar composition of electrophoretic fractions and isolated purified ML antigen was measured by the method of Dubois et al. (1956) using phenol reagent with glucose as the standard. Aminosugar was measured after hydrolysis of the samples at
1 80 w
& 60
E LO LL
30
20
1 118
L 1/32
DILU7ION OF SLRUM
FIGURE 1 - Neutralization of cytotoxic effect of antiML and anti-H-2d antisera by crude soluble 3 M KCI
extract from L1210 cells. Target cells leukaemia L1210.
WA Sera absorbed with 3 M KCI extract fro:n L1210
cells (5 mg protein/ml); 0unabsorbed sera.
t
100" C for 4 h in 4 N HCI by the method of Rissing et al. (1955) using glucosamine as the standard. Sialic acid was measured by the method of Warren (1959) after hydrolysis of the samples at 80" C for 1 h with a final concentration of 0.5 M H,SO,. RESULTS
The crude, soluble 3 M KCI extract from L 1210 leukaemia cells neutralized the cytotoxic activity from anti-ML and anti-H-2d antisera. The cytotoxic activity of ar1ti-H-2~alloserum diluted 1 :8 could be absorbed by crude, soluble 3 M KCI extract (Fig. 1) (from 1 0 0 % the cytotoxicity is decreased to 17%). Similarly, the hetero- and allo-anti-ML sera after absorption appeared to be non-cytotoxic for MLpositive leukaemia cells (Fig. 1). The preparative polyacrylamide gel electrophoresis was applied as the first step in purification of the crude extracts. The profiles showing the typical electrophoretic pattern of 3 M KCI extracts are given in Figure 2. The presence of antigen in all fractions obtained was tested by their ability to neutralize the cytotoxic effect of anti-ML and anti-H-2d sera. Fractions No. 34, 35 and 36 neutralized the cytotoxic anti-ML activity, as well as that from hetero- and alloantisera (to about 50% and, after concentration, to 20%). After absorption with the same fractions the antiH-2d allosera remained cytotoxic for ML-positive leukaemia cells. On the other hand, the a11ti-H-2~ cytotoxic activity could be neutralized by fractions No. 70-74 up to 20% (Fig. 4). As could be expected, these five fractions were not able to absorb any cytotoxicity from anti-ML sera. The selected ML-positive fractions (No. 34-36) were analyzed by polyacrylamide gel disc electrophoresis (SDS-PAGE). The results of electrophoresis were similar in all three fractions. In each instance a single, sharp band was obtained with a molecular 1
FIGURE 2 - Electrophoretic gel profile of 3 M KC1 extract from L1210 cell fractions obtained by preparative discontinuous acrylamide-gel electrophoresis (PH 9.6; 7.5% acrylamide gel). 50mg protein-eluted with PBS at constant current-20 ma, 4" C. Fractions (1.7 ml) were collected; absorbance at 280 nm. Neutralization of cytotoxic activity from anti-ML serum by fractions No. 3436 x-x, after concentration m, and anti H-2d serum by fractions N O . 70-74 A-A.
493
CHARACTERIZATION OF ML ANTIGEN
weight estimated to be 73,500 daltons from the relative migration in gel by coelectrophoresis with standard molecular weight markers. I n addition to the main band (73,500), weak bands with higher mol. wt (80,000-90,000) were observed (Fig. 3, band 3 c). Positive, but rather weak Schiff reactions of the main band (73,500) indicated the presence of carbo hydrate. The affinity chromatography method for isolation and purification of ML antigen from crude soluble 3 M KCI extract was also applied. 3 M KCI extract from L 1210cells was added to the column filled with appropriate immunoabsorbent (rabbit anti-ML globulin coupled with CNBr-activated Sepharose 4 B). As mentioned above, the crude, soluble 3 M KCI extract (Fig. 3a) contained both ML and H-2d specificities. The column was eluted to an optical density of 0.02 at 280 nm. The eluate tested for H-2d specificity in neutralization tests gave positive reactions. The absorbed ML antigen was released by Tris. HCI buffer containing 2 M MgCll and iiiimedi-
ately afterwards dialyzed against PBS for 24 h. Using the appropriate neutralization assay the presence of ML antigen was demonstrated. The ML antigen isolated by affinity chromatography neutralized the cytotoxic effect of allo- and hetero-anti-ML sera (Fig. 4). The isolated ML antigen did not neutralize the cytotoxic effect to L 1210 cells of anti H-2d serum used as a control in neutralization tests. ML antigen isolated by affinity chromatography was analyzed by polyacrylamide gel disc electrophoresis (SDS-PAGE) and the results are shown in Figure 3d. A single sharp band with mol. wt. 73,500 was obtained. The ML antigen was concentrated about 3,360fold in comparison to the crude, soluble 3 M KCI extract from leukaemia L 1210 cells. In the solubilized ML antigen the total carbohydrate, hexosamine and sialic acid contents were determined to constitute about 5 %, 2,5 % and 2% respectively. Comparing the results from the neutralization assays (Fig. 4), it was established that the absorption capacity of the isolated M L antigen (0.093 pg protein or 56 x loBdaltons) for both hetero- and alloanti-ML sera corresponds to 1 x lo6 leukaemia L 1210 cells (210pg protein). From these values it was calculated that ML antigen constitutes 0.044 % of the total cellular proteins. Taking into consideration these data and the mol. wt of the isolated ML antigen (73.5 x lo3 dalton) it could be roughly L 1210 cell contains estimated that a single 56 lo' = 7.62 x 106molecules of ML antigen. 73.5 x 1 0 3 DISCUSSION
FIGURE 3 - SDS-polyacrylamide gel electrophoresis of a crude soluble 3 M KCI extract from (a) L 1210 cells; (6) ML-positive fraction No. 34; ( c ) No. 35 and ( d ) ML antigen isolated by affinity chromatography. The antigen and fractions were dissociated by heating in the presence of 1 % SDS, 5 M urea and beta-mercaptoethanol for 2 min at 100"C. The bands were visualized by staining with Coomassie brillant blue.
Leukaemia L 1210, like other spontaneous and induced leukaemias in DBA/2 mice, has an expression of MMTV-related antigen, called mammary leukaemia (ML) antigen, since its presence can be demonstrated also on cells of mouse mammary cancer (Stuck et al., 1964; Kisielow et al., 1971; Radzikowski et al., 1972; Steuden et a!,, 1978). Screening of a variety of mouse tumours by different techniques demonstrates expression of MMTV infection only in mammary tumours, ML-positive leukaemias and Leydig cell tumours of MTVinfected mice. In contrast to mammary turnours, in which all viral structural proteins are synthesized, the synthesis of MMTV proteins is limited to two antigens in ML-positive leukaemias and Leydig tumours (Nowinski et a/., 1971). In addition, electron microscopic studies reveal that in these last tumours viral intracytoplasmic A particles are present, while there are not, as a rule, any budding virions (Nowinski et al., 1971). However, in some of the leukaemias induced in GR mice, some few extiacellular B particles could be detected in addition to large numbers of intracytoplasmic A particles (Hilgers et af., 1975; Calafat et al., 1974.) Immune cross-reactions between ML antigen and MMTV antigenic products were detected by various authors in different tests with the use of either antiMMTV antisera (Hilgers et a/., 1975; Calafat et a[., 1974; Dickson et al., 1976; Smith and Lee, 1975; Tanaka, 1977) and/or anti-ML sera as described in
494
ZAK-NEJMARK ET AL.
* NUMBER OF L1210 CELLS USED FOR ABSORPTION
11256 11128 1/6L 1/32 1/16 1/8
l/L
112
111
DILUTION OF PREPARATIONS
FIGURE 4 - Neutralization of cytotoxic activity from anti-ML sera by various numbers of L1210 cells; Crude 3 M KCl extract of L1210 cells purified by affinity chromatography ML antigen. X - - - - - x TO826 rabbit anti-ML globulin diluted 1/16 absorbed with L1210 cells; O-.-.-O C130 mouse antiC130 mouse ML serum diluted 1/14 absorbed with L1210 cells; .-..-..-. anti-ML serum diluted 1/4neutralized by 3 M KCI extract 50 mg/ml; 0 0 C130 mouse anti-ML serum diluted 1/4 neutralized by ML antigen 120 ,ug/ml.
this and our former papers (Radzikowski et al., 1977; Steuden et a1.,1978). The anti-ML cytotoxic activity can be absorbed by ML-positive leukaemia and mammary tumour cells, as well as by antigenic material isolated from intracytoplasmic A and B particles (our own, unpublished data). As described in this paper, the ML antigen isolated and purified by SDS-PAGE and affinity chromatography from leukaemia L 1210 cells has a mol. wt. of 73, 500. Using similar techniques, e.g. immunological precipitation, SDS-PAGE and rabbit anti-MMTV serum, Dickson et al. (1976) have detected that in MMTV-producing cultures of mouse mammary tumours the cells synthesize a virusrelated polypeptide of molecular weight 73,000, which is rapidly labelled during a short pulse, but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp49 and gp37.5-33.5. Cell fractionation procedure shows that these glycopeptides are present in the membrane fraction of the cytoplasm (Dickson et al., 1975, 1976). The analysis of kinetics of viral protein synthesis indicates that the cell-associated viral glycoproteins are formed by cleavage of a precursor molecule of molecular weight 73,000. The precursor has an estimated half-life of 50 to 60 min, which coincides with parallel appearance of gp49 and gp37.5-33.5. A comparison of tryptic digest maps of the glycoproteins involved supports the authors' hypothesis that both viral glycoproteins are derived from gp 73,000 (Dickson et al., 1976).
In addition to these similarities, there are some differences between the results obtained from various laboratories concerning the number, molecular weight and relative amounts of major virion polypeptides, as well as in the number of minor components detected (Dickson and Skehel, 1974; Teramoto et al., 1974; Van Blitterswijk et al., 1975; Yagi and Compans, 1977; Zotter et a1.,1976). These disparities between the various results may be attributed to differences in the source of virus, strain of host cells, in vivo or in vitro derivation, or proteolytic cleavage events which may all influence the observed polypeptide species (Yagi and Compans, 1977). ACKNOWLEDGEMENTS
The authors wish to express their thanks to Dr. E. A. Boyse, Sloan-Kettering Cancer Institute, New York, N. Y. and to Dr. A. Lengerova, Institute of Molecular Genetics, Prague, Czechoslovakia, for providing valuable experimental material and also to Dr. J. Kieler, Dr. K. Ulrich and Dr. J. Forchhammer, Fibiger Laboratory, Copenhagen, Denmark, for their valuable remarks and corrections of the manuscript. The competent and skilful assistance of Mrs. J. Topp-Radzikowska and Mrs. K. Rychlewska is gratefully acknowledged. This work was supported by Grant No. 10.5 from the Polish Academy of Sciences.
CHARACTERIZATION OF ML ANTIGEN
495
ANTIGENE D E LA LEUCEMIE ET DES TUMEURS -MAMMAIRES (ML) ISOLE A PARTIR D E CELLULES D E LEUCEMIE L 1210 Les extraits bruts au KC l 3 M de cellules L 1210 et I’antigene ML purifie ont ete caracterisks et la presence de l’antigene ML a et6 verifiee & l’aide de divers serums anti-ML, et d’antiserums contre les particules B du MMTV tclatkes. Certaines fractions ML-positives obtenues par electrophorese preparative en gel de polyacrylamide et des antigenes ML purifies, isoles par chromatographie d’afinite, ont ete analyses en electrophorese en gel de polyacrylamide (SDS-PAGE). On a obtenu la meme bande avec les deux precedes. Le poids moleculaire estime est de 73 000 pour les deux preparations. Les auteurs examinent la relation entre l’antigbne ML, les produits antigeniques du MMTV et les proteines virales associees A la cellule.
REFERENCES
CALAFAT, J., BUIJS,F., and HAGEMAN, Ph., Distribution of J. Mouriquand (ed.), INSERM Colloquium, Fundamental Research on Mammary Tumours, pp. 137-146, INSERM, virus particles and mammary tumour virus antigens in Grenoble (1972). mammary turnours, transformed BALB/c mouse kidney cells and G R ascites leukaemia cells. J. nut. Cancer Inst., 53, RADZIKOWSKI, Cz., ZAK-NEJMARK; T., WYSOCKA, M., and 977-991 (1974). STEUDEN,J., Production of anti-ML antibodies in the xenogeneic host. Arch. immun. ther. Exp., 25, 507-513 (1977). DICKSON,C., PUMA,J. P., and NANDI,S., Intracellular synthesis of mouse mammary tumour virus polypeptides: REISFELD, R. A ,, PELLEGRINO, M. A., and KAHAN,B. D., Salt indication of a precursor glycoprotein. J. Virol., 16, 250-258 extraction of soluble HL-A antigen. Science, 172, 1 1 34-1 136 (1 975). (1971). DICKSON,C . , PUMA,J. P., and NANDI,S., Identification of RISSING,J. L., STROMINGER, J. L., and LELOIR,L. F.. A precursor protein to the major glycoproteins of mouse modified colorimetric method for the estimation of Nmammary tumour virus. 1. Virol., 17, 275-282 (1976). acetyloamino sugars. J. biol. Chem., 217, 959-966 (1955). DICKSON, C., and SKEHEL, J. J., The polypeptide composition RODBARD, D., and CHRAMBACH, A., Estimation of molecular of mouse mammary tumour virus. Virology, 58, 387-395 radius, free mobility and valence using polyacrylamide gel (1974). electrophoresis. Anal. Biochem., 40, 95-134 (1971). DUBOIS, M., GILLES,K. A,, HAMILTON, J. K., REBERS, P. A., SARKAR,N. H.,NOWINSKI,R. C., and MOORE,D. H., and SMITH,F., Colorimetric methods for determination of Characteristic of the structural components of the mouse sugars and related substances. Analyt. Chem., 28, 350-356 mammary tumour virus : morphological and biochemical (1956). studies. Virology, 46, 1-20 (1971). GORER,P. A,, and O’GORMAN, P.,The cytotoxic activity of SMITH,G. H., and LEE,B. K., Brief communication: mouse isoantibodies in mice. Transplant. Bull., 3, 142-143 (1956). mammary tumour polypeptide precursor in intracytoplasmic HILGERS, J., HAVERMAN, J., NUSSE,R . , VAN B L I T T E R S ~ J K ,“ A ” particles. J. nut. Cancer Inst., 55, 493-496 (1975). Ph., VAN NIE, R., and W. J., CLETON:F. J., HAGEMAN, STEUDEN,J., RADZIKOWSKI, Cz., and ~ A K - N E J M A RT., K, CALAFAT, J., Immunologic, virologic and genetic aspects of Serological identification of viral and viral related antigens mammary tuniour virus induced cell surface antigens : o n DBA/2 mouse leukaemia lymphocytes. Arch. immun. presence of these antigens and Thy 1.2. antigen on murine ther. Exp., in press (1978). mammary gland and tumour cells. J. nat. Cancer Inst., 54, STUCK,D., BOYSE,E. A., OLD, L. J., and CARSWELL, E. A., 1323-1333 (1975). ML: a new antigen found in leukaemias and mammary HILGERS,J., THEUNS,G. J., and VAN NIE, R., Mammary turnours of the mouse. Nature (Lond.), 203,1033-1034 (1964). tumour virus (MTV) antigens in normal and mammary TANAKA, H., Precursor product relationship between nontumour bearing mice. Int. J. Cancer, 12, 568-576 (1973). glycosylated polypeptides of “ A ” and “ B ” particles of JANSEN: J. L. J., KOENE,R. A. P., KAMP,G. J.. TAMBOER,mouse mammary tuniour virus. Virology, 76, 835-850 (1977). W. P. M., and WIJDEVELD, P. G. A. B., Isolation of pure IgG TERAMATO. Y. A., PUENTERS,M. J., YOUNG,J. T., and subclasses from mouse alloantiserum and their activity in CARDIFF, R. D., Structure of the mouse mammary tumour enhancement and hyperacute rejection of skin. J. Immunol., virus: polypeptides and glycoproteins. J. Virol., 13, 41 1-418 115, 387-391 (1975). (I 974). KISIELOW, P., RADZIKOWSKI, Cz., STEUDEN, J., SZKUDLAREK, VAN BLITTERSWUK, W. J., EMMELOT,P., HILGERS,J., J., and KRAwczYNsKi, K . , Mammary tumour virus associated KAMLAG, D., NUSSE,R., and FELTKAMP, C. A., Quantitation antigenicity of L 1210 mouse leukaemia cells. Arch. immun. of virus induced (MLr) and normal (Thy l.2), cell surface ther. Exp., 19, 789-799 (1971). antigens in isolated plasma membranes and the extracellularLOWRY,0. H., ROSENBROUGH, N. J., FARR, A. L. and ascites fluid of mouse leukaemia cells. Cancer Res., 35, 2743RANDALL, R. J.. Protein measurement with the Folin phenol 2751 (1975). reagent. J. biol. Chem., 193, 265-275 (1951). WARREN,L., The thiobarbituric acid assay of sialic acid. NOWINSKI, R. C., OLD, L. J., MOORE,D. H., GOERING, G., J. biol. Chem., 234, 1971-1975 (1959). and BOYSE,E. A., A soluble antigen of the mammary tumour WEBER,K., and OSBORN,M., The reliability of molecular virus. Virology, 34, 1-14 (1967). weight determinations by dodecylsulphate-polyacrylamide NOWINSKI, R. C., SARKAR, N. H ,OLD,L. J., MOORE,D. H., gel electrophoresis. J. biol. Chem., 244, 4406-4411 (1969). SCHEER, D. J., and HILGERS,J., Characteristic of the strucYAGI,M. J., and COMPANS, R. W., Structural components of tural components of the mouse mammary tumour virus: viral mouse mammary tumour virus. I. Polypeptides of the virion. proteins and antigens. Virology, 46, 21-38 (1971). Virology, 76, 751-766 (1977). OYAMA, V. I., and EAGLE,H., Measurement of cell growth in ZOTTER, St., KRYUKOWA, I. N., BUKRINSKAYA, A. G., tissue culture with phenol reagent (Folin-Ciocalteau 22245). LEZHNEVA, 0. M., ILYIN,K. V., MILLER,M., and MILLER, Proc. Soc. exp. Biol. ( N . Y . ) , 91, 305-307 (1956). G. G., Presence of the p 27 antigenicity and absence of the gp 52 antigenicity and leukaemia virus antigen in intraRADZIKOWSKI, Cz., KISIELOW, P., ZAK,T.,and INGLOT, A. D., cytoplasmic ‘‘ A ” particles (iAp) of mouse mammary tumour Antigenic expression of MMTV and MuLV in DBA/2 virus origin. Arch. Geschwulstforsch., 46, 621-629 (1976). leukaemia and DBA/2 and C3H mammary tumour cells. In:
MAMMARY LEUKAEMlA (ML) ANTIGEN ISOLATED FROM L 1210 LEUKAEMIA CELLS Teresa ZAK-NEJMARK, Jadwiga STEUDEN and Czeslaw RADZIKOWSKI Department of Tumour Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
Crude 3 M KCI extracts from L 1210 cells and purified M L antigen were characterized and the presence of M L antigens was verified by various antiM L sera, as well as by antisera to disrupted M M T V B particles. Selected ML-positive fractions from preparative polyacrylamide electrophoresis and purified M L antigen isolated by affinity chromatography were analyzed by polyacrylamide disc electrophoresis. W i t h both procedures the same sharp band was obtained. The molecular weight of both preparations was found t o be 73,500 as estimated. The relationship between M L antigen, M M T V antigenic products and cell-associated viral proteins i s discussed.
The mammary leukaemia (ML) antigen is the mouse mammary tumour virus (MMTV)-related cellular membrane antigen characteristic of cells of leukaemias and mammary cancers induced or occurring naturally in DBAJ2, C3H, A and GR mice. This antigen was discovered by Stuck et al. in a direct complement-dependent cytotoxic test with the use of alloantisera produced in C57BL/6 mice by immunization with DBA/2 leukaemia cells (Stuck et al., 1964). In our previous paper (Steuden et al., 1978) we demonstrated the presence of ML antigen on leukaemia L 1210 cells by using both heterologous (rabbit) and alloantisera (BALB/c) and anti-ML sera produced in syngeneic DBA/2 mice. In addition, by applying appropriate absorptions and blocking techniques combined with ferritin-labelled antibodies in electron microscopy studies (EM), we demonstrated the independent localization of ML antigen and murine leukaemia virus-Gross (MuLV-G) cellular antigen on L 1210 cells (Steuden et al., 1978). The ferritin-labelled antigenic sites appeared to be free from budding viral particles. It should be mentioned that in our EM studies no MMTV B particles but abundant intracytoplasmic A particles (B particle precursors) and MuLV C particles could be demonstrated (Radzikowski et al., 1972). An interrelationship and/or identity between ML and MMTV antigenic products is emphasized by several authors. Positive serological reactions with ML-positive cells were observed not only with antiML sera but also with antisera obtained by immunization of rabbits with isolated, ether-disrupted and purified B particles (Nowinski et al., 1967, Hilgers et al., 1975; Van Blitterswijk et al., 1975; Sarkar et a/., 1971). There is, however, no convincing evidence available that antisera obtained in syngeneic, allogeneic and xenogeneic hosts identify the same antigen or antigenic complex on leukaemic cells.
Thus, it seems appropriate, as suggested by Hilgers et al. (1975), to use the name ‘‘ MLm ” for the antigen identified by antisera produced in mice and “ MLr ” for the antigen identified by heterologous (rabbit) antisera against MMTV B particles. In the present studies the 3~ KCI extracts of leukaemia cells purified by polyacrylamide gel electrophoresis (PAGE) and the antigen from leukaemia L 1210 cells purified by affinity chromatography were characterized and their identity verified by anti-ML sera, obtained from both allogeneic and xenogeneic hosts. Heterologous antisera against disrupted MMTV B particles were also used (Steuden et al., 1978). The use of these latter antisera seems especially valuable, if one considers that the DBA/2 leukaemia L 1210, contrary to the GR leukaemia GRSL/2 (Hilgers et al., 1973, 1975) was found to produce no budding MMTV B particles. The positive serological membrane reactions of L 1210 cells with anti-MMTV disrupted B particle antisera could occur only if MMTV antigenic products were present on the cell surface. MATERIAL A N D METHODS
Animals Mice of all strains used were maintained by brother xsister mating at the Inbred Mice Center of the Institute of Immunology and Experimental Therapy in Wroclaw. Their main characteristics were: DBA/2 (H-2d) and C3H (H-2k) with high natural incidence of mammary tumours. BALB/c (H-2d), C57BL/6 (H-2b) with low natural tumour incidence, AKR (H-2k) with high natural incidence of leukaemia. Rabbits were obtained from the breeding colony maintained at the same Institute. Tumours L 1210 V. This transplantable DBA/2 leukaemia has been maintained at the Department of Tumour Immunology since 1969 as an ascites and a solid transplantable line. The presence of ML antigen on leukaemia cells and production of a large number of intracytoplasmic viral A particles and MuLV C particles are constant traits of these cells. We have never detected any MMTV B particles, either budding or extracellular. The cells from either ascites or leukaemic spleens were harvested, as a rule, 4 days after inoculation of 5-10 x lo6 cells intraperitoneally, or sometimes, subcutaneously. Leukaemia L 1210 cells were used for preparation of 3 M KCI extracts and purified antigenic material.
Received: December 21, 1977.
CHARACTERIZATION OF ML ANTIGEN
E 0" G2. This transplantable C57BL/6 lymphoma, maintained at our Department as a transplantable line since 1976, was received from Dr. E. A. Boyse, Sloan-Kettering Cancer Institute, New York, N. Y. The presence of Gross cellular surface antigen (GCSA) as well as of other MuLV proteins, is a constant feature of the leukaemia cells. This leukaemia line and cells from naturally occurring AKR leukaemias were used as GCSA-positive reference lines for absorption of anti-MuLV-Gross antibodies from oligovalent antisera produced by immunization with leukaemia L 1210 cells. Cells from naturally occuring mammary tumours in C3H and DBA/2 mice were used as ML-positive reference lines for absorption or as controls in cytotoxic tests. Antisera Alloantisera against ML antigen (anti-ML sera) were prepared by immunization of BALB/c (H-2d) mice with leukaemia L 1210 (H-2d) cells. These sera reacted in cytotoxicity tests with ML-positive cells of spontaneous mammary tumours of DBA/2 and C3H mice and L 1210 leukaemia, but were not toxic to normal DBA/2 lymphoid cells. Cytotoxic anti-ML antibodies could be removed by absorption with MLpositive cells of spontaneous or transplantable mammary tumours and/or of leukaemia L 1210. The anti-ML sera did not react in cytotoxicity tests with leukaemia cells of Gross-positive spontaneous AKR lymphomas or transplantable E 0' G2 leukaemia in C57BL/6 mice. The anti-ML cytotoxic activity could not be removed by absorption with Gross-positive leukaemia cells. Immunization schedules and serological characterization of these sera are described elsewhere (Steuden et al., 1977). Anti-H-2d serum (C57BL/6 anti-BALB/c) was kindly provided by Dr. A. Lengerova of the Institute of Molecular Genetics, Prague. Heterologous anti-ML sera were produced in rabbits by immunization with leukaemia L 1210 cells as described elsewhere (Radzikowski et al., 1977).After repeated absorption with normal DBA/2 cells, these sera reacted in cytotoxic tests exclusively with ML-positive cells. Normal DBA/2 lymphoid cells and Gross-positive AKR and Eo" G2 leukaemia czlls were used as negative controls. Soluble antigen preparation The preparation of 3 M KCI extracts was a modification of the method used by Reisfeld et al. (1971) for solubilization of transplantation antigens. Leukaemia L 1210 cells were washed twice with cold phosphate-buffered saline (PBS, PH 7.4). Washed cells were resuspended in cold 3 M KCI in PBS at a concentration of los cells in 20 ml and gently agitated for 16 h at 4" C. After centrifugation at 165,OOOg for 1 h, the supernatant was separated from the viscous pellet and dialyzed against 200-fold volumes of cold PBS for 48 h with three changes. The dialyzate was clarified by centrifugation at 150,000g for 20 min and concentrated by ultrafiltration. Crude soluble antigen preparations were stored at -20" C and used for preparative polyacrylamide gel electrophoresis and affinity chromatography.
49 1
Preparative polyacrylamide gel electrophoresis Preparative, discontinuous polyacrylamide gel electrophoresis was used to purify the antigenic principle. Electrophoresis was performed in buffer system B of Rodbard and Chrambach (1971) at 4" C at constant current 20mA. A sample of 50mg protein was applied to the gel. After elution with PBS, fractions of 1.7 ml each were collected when the tracking dye (0.05 % Coomassie brilliant blue) had reached the end of the gel. Protein was determined in all fractions and total carbohydrate, aminosugar and sialic acid were determined in selected fractions. Absorption capacity of all fractions was tested in cytotoxic neutralization assay. Polyacrylamide gel disc electrophoresis The samples of crude soluble antigen were mixed with urea, SDS and mercaptoethanol at a final concentration of 8 M, 0.1 % and 2 % respectively. The mixture was heated to 100" C for 2 min and applied directly to the cooled gel. Samples were electrophoresed in the presence of SDS as described by Weber and Osborn (1969). Gels were stained with Coomassie brilliant blue and periodic acid-Schiff (PAS). Molecular weight of the ML antigen was estimated from its relative migration in gels by coelectrophoresis with standard molecular weight markers: cytochrome C (12,400), horse myoglobin (1 7,8OO), bovine serum albumin (67,000) (Serva, Heidelberg, West Germany), trypsin (23,300), ovalbumin (43,000) (Sigma, St. Louis, USA) and galactosidase (1 30,000) (Biochemicals, Wisconsin, USA). Molecular weights were calculated according to Weber and Osborn (1969) by the following formula: Distance of Drotein Length - before migration staining Mobility = X Length after Distance of dye destaining migration The mobilities were plotted against the known molecular weight expressed on a semi-logarithmic scale. Afinity chromatography Immunoabsorbent was prepared by the technique of Jansen etal. (1975). A column (K9/15 Pharmacia, Uppsala, Sweden) of CNBr-activated Sepharose 4B (Pharmacia) coupled with rabbit anti-ML globulins was used. The solution containing the ML antigen (crude 3 M KCI extracts from L 1210 leukaemia cells) was applied to the column at the rate of 20 ml/h. Absorbed antigen was released by , 7.6) containing 2 M Tris-HCI buffer (50 m ~ PH MgClz and immediately afterwards dialyzed against PBS for 24 h. The dialyzed eluate was concentrated by vacuum ultrafiltration up to 0.1 mg protein per 1 ml. Protein estimation The method of Lowry et al. (1951) was used with bovine serum albumin as a standard or the total concentration of protein in the cell suspension was determined according to the method of Oyama and Eagle (1956).
492
ZAK-NEJMARK ET AL.
Neutralization assay for soluble antigen absorption capacity The neutralization of anti-ML cytotoxic activity from allo- and heterosera was performed as follows : serial dilutions of anti-ML sera were incubated with successive electrophoretic fractions of purified antigenic preparations and in a parallel experiment with various numbers of L 1210 cells for 1 h at 37" C. Then complement-dependent cytotoxic tests according to the method of Gorer and O'Gorman (1956) were performed. Sugar composition Sugar composition of electrophoretic fractions and isolated purified ML antigen was measured by the method of Dubois et al. (1956) using phenol reagent with glucose as the standard. Aminosugar was measured after hydrolysis of the samples at
1 80 w
& 60
E LO LL
30
20
1 118
L 1/32
DILU7ION OF SLRUM
FIGURE 1 - Neutralization of cytotoxic effect of antiML and anti-H-2d antisera by crude soluble 3 M KCI
extract from L1210 cells. Target cells leukaemia L1210.
WA Sera absorbed with 3 M KCI extract fro:n L1210
cells (5 mg protein/ml); 0unabsorbed sera.
t
100" C for 4 h in 4 N HCI by the method of Rissing et al. (1955) using glucosamine as the standard. Sialic acid was measured by the method of Warren (1959) after hydrolysis of the samples at 80" C for 1 h with a final concentration of 0.5 M H,SO,. RESULTS
The crude, soluble 3 M KCI extract from L 1210 leukaemia cells neutralized the cytotoxic activity from anti-ML and anti-H-2d antisera. The cytotoxic activity of ar1ti-H-2~alloserum diluted 1 :8 could be absorbed by crude, soluble 3 M KCI extract (Fig. 1) (from 1 0 0 % the cytotoxicity is decreased to 17%). Similarly, the hetero- and allo-anti-ML sera after absorption appeared to be non-cytotoxic for MLpositive leukaemia cells (Fig. 1). The preparative polyacrylamide gel electrophoresis was applied as the first step in purification of the crude extracts. The profiles showing the typical electrophoretic pattern of 3 M KCI extracts are given in Figure 2. The presence of antigen in all fractions obtained was tested by their ability to neutralize the cytotoxic effect of anti-ML and anti-H-2d sera. Fractions No. 34, 35 and 36 neutralized the cytotoxic anti-ML activity, as well as that from hetero- and alloantisera (to about 50% and, after concentration, to 20%). After absorption with the same fractions the antiH-2d allosera remained cytotoxic for ML-positive leukaemia cells. On the other hand, the a11ti-H-2~ cytotoxic activity could be neutralized by fractions No. 70-74 up to 20% (Fig. 4). As could be expected, these five fractions were not able to absorb any cytotoxicity from anti-ML sera. The selected ML-positive fractions (No. 34-36) were analyzed by polyacrylamide gel disc electrophoresis (SDS-PAGE). The results of electrophoresis were similar in all three fractions. In each instance a single, sharp band was obtained with a molecular 1
FIGURE 2 - Electrophoretic gel profile of 3 M KC1 extract from L1210 cell fractions obtained by preparative discontinuous acrylamide-gel electrophoresis (PH 9.6; 7.5% acrylamide gel). 50mg protein-eluted with PBS at constant current-20 ma, 4" C. Fractions (1.7 ml) were collected; absorbance at 280 nm. Neutralization of cytotoxic activity from anti-ML serum by fractions No. 3436 x-x, after concentration m, and anti H-2d serum by fractions N O . 70-74 A-A.
493
CHARACTERIZATION OF ML ANTIGEN
weight estimated to be 73,500 daltons from the relative migration in gel by coelectrophoresis with standard molecular weight markers. I n addition to the main band (73,500), weak bands with higher mol. wt (80,000-90,000) were observed (Fig. 3, band 3 c). Positive, but rather weak Schiff reactions of the main band (73,500) indicated the presence of carbo hydrate. The affinity chromatography method for isolation and purification of ML antigen from crude soluble 3 M KCI extract was also applied. 3 M KCI extract from L 1210cells was added to the column filled with appropriate immunoabsorbent (rabbit anti-ML globulin coupled with CNBr-activated Sepharose 4 B). As mentioned above, the crude, soluble 3 M KCI extract (Fig. 3a) contained both ML and H-2d specificities. The column was eluted to an optical density of 0.02 at 280 nm. The eluate tested for H-2d specificity in neutralization tests gave positive reactions. The absorbed ML antigen was released by Tris. HCI buffer containing 2 M MgCll and iiiimedi-
ately afterwards dialyzed against PBS for 24 h. Using the appropriate neutralization assay the presence of ML antigen was demonstrated. The ML antigen isolated by affinity chromatography neutralized the cytotoxic effect of allo- and hetero-anti-ML sera (Fig. 4). The isolated ML antigen did not neutralize the cytotoxic effect to L 1210 cells of anti H-2d serum used as a control in neutralization tests. ML antigen isolated by affinity chromatography was analyzed by polyacrylamide gel disc electrophoresis (SDS-PAGE) and the results are shown in Figure 3d. A single sharp band with mol. wt. 73,500 was obtained. The ML antigen was concentrated about 3,360fold in comparison to the crude, soluble 3 M KCI extract from leukaemia L 1210 cells. In the solubilized ML antigen the total carbohydrate, hexosamine and sialic acid contents were determined to constitute about 5 %, 2,5 % and 2% respectively. Comparing the results from the neutralization assays (Fig. 4), it was established that the absorption capacity of the isolated M L antigen (0.093 pg protein or 56 x loBdaltons) for both hetero- and alloanti-ML sera corresponds to 1 x lo6 leukaemia L 1210 cells (210pg protein). From these values it was calculated that ML antigen constitutes 0.044 % of the total cellular proteins. Taking into consideration these data and the mol. wt of the isolated ML antigen (73.5 x lo3 dalton) it could be roughly L 1210 cell contains estimated that a single 56 lo' = 7.62 x 106molecules of ML antigen. 73.5 x 1 0 3 DISCUSSION
FIGURE 3 - SDS-polyacrylamide gel electrophoresis of a crude soluble 3 M KCI extract from (a) L 1210 cells; (6) ML-positive fraction No. 34; ( c ) No. 35 and ( d ) ML antigen isolated by affinity chromatography. The antigen and fractions were dissociated by heating in the presence of 1 % SDS, 5 M urea and beta-mercaptoethanol for 2 min at 100"C. The bands were visualized by staining with Coomassie brillant blue.
Leukaemia L 1210, like other spontaneous and induced leukaemias in DBA/2 mice, has an expression of MMTV-related antigen, called mammary leukaemia (ML) antigen, since its presence can be demonstrated also on cells of mouse mammary cancer (Stuck et al., 1964; Kisielow et al., 1971; Radzikowski et al., 1972; Steuden et a!,, 1978). Screening of a variety of mouse tumours by different techniques demonstrates expression of MMTV infection only in mammary tumours, ML-positive leukaemias and Leydig cell tumours of MTVinfected mice. In contrast to mammary turnours, in which all viral structural proteins are synthesized, the synthesis of MMTV proteins is limited to two antigens in ML-positive leukaemias and Leydig tumours (Nowinski et a/., 1971). In addition, electron microscopic studies reveal that in these last tumours viral intracytoplasmic A particles are present, while there are not, as a rule, any budding virions (Nowinski et al., 1971). However, in some of the leukaemias induced in GR mice, some few extiacellular B particles could be detected in addition to large numbers of intracytoplasmic A particles (Hilgers et af., 1975; Calafat et al., 1974.) Immune cross-reactions between ML antigen and MMTV antigenic products were detected by various authors in different tests with the use of either antiMMTV antisera (Hilgers et a/., 1975; Calafat et a[., 1974; Dickson et al., 1976; Smith and Lee, 1975; Tanaka, 1977) and/or anti-ML sera as described in
494
ZAK-NEJMARK ET AL.
* NUMBER OF L1210 CELLS USED FOR ABSORPTION
11256 11128 1/6L 1/32 1/16 1/8
l/L
112
111
DILUTION OF PREPARATIONS
FIGURE 4 - Neutralization of cytotoxic activity from anti-ML sera by various numbers of L1210 cells; Crude 3 M KCl extract of L1210 cells purified by affinity chromatography ML antigen. X - - - - - x TO826 rabbit anti-ML globulin diluted 1/16 absorbed with L1210 cells; O-.-.-O C130 mouse antiC130 mouse ML serum diluted 1/14 absorbed with L1210 cells; .-..-..-. anti-ML serum diluted 1/4neutralized by 3 M KCI extract 50 mg/ml; 0 0 C130 mouse anti-ML serum diluted 1/4 neutralized by ML antigen 120 ,ug/ml.
this and our former papers (Radzikowski et al., 1977; Steuden et a1.,1978). The anti-ML cytotoxic activity can be absorbed by ML-positive leukaemia and mammary tumour cells, as well as by antigenic material isolated from intracytoplasmic A and B particles (our own, unpublished data). As described in this paper, the ML antigen isolated and purified by SDS-PAGE and affinity chromatography from leukaemia L 1210 cells has a mol. wt. of 73, 500. Using similar techniques, e.g. immunological precipitation, SDS-PAGE and rabbit anti-MMTV serum, Dickson et al. (1976) have detected that in MMTV-producing cultures of mouse mammary tumours the cells synthesize a virusrelated polypeptide of molecular weight 73,000, which is rapidly labelled during a short pulse, but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp49 and gp37.5-33.5. Cell fractionation procedure shows that these glycopeptides are present in the membrane fraction of the cytoplasm (Dickson et al., 1975, 1976). The analysis of kinetics of viral protein synthesis indicates that the cell-associated viral glycoproteins are formed by cleavage of a precursor molecule of molecular weight 73,000. The precursor has an estimated half-life of 50 to 60 min, which coincides with parallel appearance of gp49 and gp37.5-33.5. A comparison of tryptic digest maps of the glycoproteins involved supports the authors' hypothesis that both viral glycoproteins are derived from gp 73,000 (Dickson et al., 1976).
In addition to these similarities, there are some differences between the results obtained from various laboratories concerning the number, molecular weight and relative amounts of major virion polypeptides, as well as in the number of minor components detected (Dickson and Skehel, 1974; Teramoto et al., 1974; Van Blitterswijk et al., 1975; Yagi and Compans, 1977; Zotter et a1.,1976). These disparities between the various results may be attributed to differences in the source of virus, strain of host cells, in vivo or in vitro derivation, or proteolytic cleavage events which may all influence the observed polypeptide species (Yagi and Compans, 1977). ACKNOWLEDGEMENTS
The authors wish to express their thanks to Dr. E. A. Boyse, Sloan-Kettering Cancer Institute, New York, N. Y. and to Dr. A. Lengerova, Institute of Molecular Genetics, Prague, Czechoslovakia, for providing valuable experimental material and also to Dr. J. Kieler, Dr. K. Ulrich and Dr. J. Forchhammer, Fibiger Laboratory, Copenhagen, Denmark, for their valuable remarks and corrections of the manuscript. The competent and skilful assistance of Mrs. J. Topp-Radzikowska and Mrs. K. Rychlewska is gratefully acknowledged. This work was supported by Grant No. 10.5 from the Polish Academy of Sciences.
CHARACTERIZATION OF ML ANTIGEN
495
ANTIGENE D E LA LEUCEMIE ET DES TUMEURS -MAMMAIRES (ML) ISOLE A PARTIR D E CELLULES D E LEUCEMIE L 1210 Les extraits bruts au KC l 3 M de cellules L 1210 et I’antigene ML purifie ont ete caracterisks et la presence de l’antigene ML a et6 verifiee & l’aide de divers serums anti-ML, et d’antiserums contre les particules B du MMTV tclatkes. Certaines fractions ML-positives obtenues par electrophorese preparative en gel de polyacrylamide et des antigenes ML purifies, isoles par chromatographie d’afinite, ont ete analyses en electrophorese en gel de polyacrylamide (SDS-PAGE). On a obtenu la meme bande avec les deux precedes. Le poids moleculaire estime est de 73 000 pour les deux preparations. Les auteurs examinent la relation entre l’antigbne ML, les produits antigeniques du MMTV et les proteines virales associees A la cellule.
REFERENCES
CALAFAT, J., BUIJS,F., and HAGEMAN, Ph., Distribution of J. Mouriquand (ed.), INSERM Colloquium, Fundamental Research on Mammary Tumours, pp. 137-146, INSERM, virus particles and mammary tumour virus antigens in Grenoble (1972). mammary turnours, transformed BALB/c mouse kidney cells and G R ascites leukaemia cells. J. nut. Cancer Inst., 53, RADZIKOWSKI, Cz., ZAK-NEJMARK; T., WYSOCKA, M., and 977-991 (1974). STEUDEN,J., Production of anti-ML antibodies in the xenogeneic host. Arch. immun. ther. Exp., 25, 507-513 (1977). DICKSON,C., PUMA,J. P., and NANDI,S., Intracellular synthesis of mouse mammary tumour virus polypeptides: REISFELD, R. A ,, PELLEGRINO, M. A., and KAHAN,B. D., Salt indication of a precursor glycoprotein. J. Virol., 16, 250-258 extraction of soluble HL-A antigen. Science, 172, 1 1 34-1 136 (1 975). (1971). DICKSON,C . , PUMA,J. P., and NANDI,S., Identification of RISSING,J. L., STROMINGER, J. L., and LELOIR,L. F.. A precursor protein to the major glycoproteins of mouse modified colorimetric method for the estimation of Nmammary tumour virus. 1. Virol., 17, 275-282 (1976). acetyloamino sugars. J. biol. Chem., 217, 959-966 (1955). DICKSON, C., and SKEHEL, J. J., The polypeptide composition RODBARD, D., and CHRAMBACH, A., Estimation of molecular of mouse mammary tumour virus. Virology, 58, 387-395 radius, free mobility and valence using polyacrylamide gel (1974). electrophoresis. Anal. Biochem., 40, 95-134 (1971). DUBOIS, M., GILLES,K. A,, HAMILTON, J. K., REBERS, P. A., SARKAR,N. H.,NOWINSKI,R. C., and MOORE,D. H., and SMITH,F., Colorimetric methods for determination of Characteristic of the structural components of the mouse sugars and related substances. Analyt. Chem., 28, 350-356 mammary tumour virus : morphological and biochemical (1956). studies. Virology, 46, 1-20 (1971). GORER,P. A,, and O’GORMAN, P.,The cytotoxic activity of SMITH,G. H., and LEE,B. K., Brief communication: mouse isoantibodies in mice. Transplant. Bull., 3, 142-143 (1956). mammary tumour polypeptide precursor in intracytoplasmic HILGERS, J., HAVERMAN, J., NUSSE,R . , VAN B L I T T E R S ~ J K ,“ A ” particles. J. nut. Cancer Inst., 55, 493-496 (1975). Ph., VAN NIE, R., and W. J., CLETON:F. J., HAGEMAN, STEUDEN,J., RADZIKOWSKI, Cz., and ~ A K - N E J M A RT., K, CALAFAT, J., Immunologic, virologic and genetic aspects of Serological identification of viral and viral related antigens mammary tuniour virus induced cell surface antigens : o n DBA/2 mouse leukaemia lymphocytes. Arch. immun. presence of these antigens and Thy 1.2. antigen on murine ther. Exp., in press (1978). mammary gland and tumour cells. J. nat. Cancer Inst., 54, STUCK,D., BOYSE,E. A., OLD, L. J., and CARSWELL, E. A., 1323-1333 (1975). ML: a new antigen found in leukaemias and mammary HILGERS,J., THEUNS,G. J., and VAN NIE, R., Mammary turnours of the mouse. Nature (Lond.), 203,1033-1034 (1964). tumour virus (MTV) antigens in normal and mammary TANAKA, H., Precursor product relationship between nontumour bearing mice. Int. J. Cancer, 12, 568-576 (1973). glycosylated polypeptides of “ A ” and “ B ” particles of JANSEN: J. L. J., KOENE,R. A. P., KAMP,G. J.. TAMBOER,mouse mammary tuniour virus. Virology, 76, 835-850 (1977). W. P. M., and WIJDEVELD, P. G. A. B., Isolation of pure IgG TERAMATO. Y. A., PUENTERS,M. J., YOUNG,J. T., and subclasses from mouse alloantiserum and their activity in CARDIFF, R. D., Structure of the mouse mammary tumour enhancement and hyperacute rejection of skin. J. Immunol., virus: polypeptides and glycoproteins. J. Virol., 13, 41 1-418 115, 387-391 (1975). (I 974). KISIELOW, P., RADZIKOWSKI, Cz., STEUDEN, J., SZKUDLAREK, VAN BLITTERSWUK, W. J., EMMELOT,P., HILGERS,J., J., and KRAwczYNsKi, K . , Mammary tumour virus associated KAMLAG, D., NUSSE,R., and FELTKAMP, C. A., Quantitation antigenicity of L 1210 mouse leukaemia cells. Arch. immun. of virus induced (MLr) and normal (Thy l.2), cell surface ther. Exp., 19, 789-799 (1971). antigens in isolated plasma membranes and the extracellularLOWRY,0. H., ROSENBROUGH, N. J., FARR, A. L. and ascites fluid of mouse leukaemia cells. Cancer Res., 35, 2743RANDALL, R. J.. Protein measurement with the Folin phenol 2751 (1975). reagent. J. biol. Chem., 193, 265-275 (1951). WARREN,L., The thiobarbituric acid assay of sialic acid. NOWINSKI, R. C., OLD, L. J., MOORE,D. H., GOERING, G., J. biol. Chem., 234, 1971-1975 (1959). and BOYSE,E. A., A soluble antigen of the mammary tumour WEBER,K., and OSBORN,M., The reliability of molecular virus. Virology, 34, 1-14 (1967). weight determinations by dodecylsulphate-polyacrylamide NOWINSKI, R. C., SARKAR, N. H ,OLD,L. J., MOORE,D. H., gel electrophoresis. J. biol. Chem., 244, 4406-4411 (1969). SCHEER, D. J., and HILGERS,J., Characteristic of the strucYAGI,M. J., and COMPANS, R. W., Structural components of tural components of the mouse mammary tumour virus: viral mouse mammary tumour virus. I. Polypeptides of the virion. proteins and antigens. Virology, 46, 21-38 (1971). Virology, 76, 751-766 (1977). OYAMA, V. I., and EAGLE,H., Measurement of cell growth in ZOTTER, St., KRYUKOWA, I. N., BUKRINSKAYA, A. G., tissue culture with phenol reagent (Folin-Ciocalteau 22245). LEZHNEVA, 0. M., ILYIN,K. V., MILLER,M., and MILLER, Proc. Soc. exp. Biol. ( N . Y . ) , 91, 305-307 (1956). G. G., Presence of the p 27 antigenicity and absence of the gp 52 antigenicity and leukaemia virus antigen in intraRADZIKOWSKI, Cz., KISIELOW, P., ZAK,T.,and INGLOT, A. D., cytoplasmic ‘‘ A ” particles (iAp) of mouse mammary tumour Antigenic expression of MMTV and MuLV in DBA/2 virus origin. Arch. Geschwulstforsch., 46, 621-629 (1976). leukaemia and DBA/2 and C3H mammary tumour cells. In:
