Cell Biology
International
Phenotypic
Reports,
modification lymphoid
Vol. 2, No. 1, 1978
of SV40-transformed cells -in vivo
hamster
Peck-Sun Lin*, Catherine E. Butterfield, and Donald F. H. Wallach Tufts-New England Medical Center Therapeutic Radiology Department Radiobiology Division 171 Harrison Avenue Boston, Mass. 02111 * To whom correspondenc:e
should be addressed
ABSTRACT Explants of simian virus 40 (SV40)-induced lymphoid tumors yield SV40-T-antigen-positive derivatives that differ from GD248 lymphocytes propagated in suspension culture, (or -in vivo), in the substrates following respects: polygonal shape, adhesion to culture phagocytic capacity, lack of immunoglobulin and a chromo--in vitro, When GDsome complement at least twice that of GD248 lymphocytes. 248 lymphocytes are propagated as suspension in vitro, no such adherent variants can be detected. However, sequential in vivo pas-__ sage of GD248 lymphocytes obtained Erom the suspension-culture lines also yield adherent cell lines upon explantation in vitro-In-jection of adherent cells into hamsters produces tumors with histological features of reticulum cell ,sarcoma. Injection of Simian Virus 40 (SV40) into three-week old outbred Syrian golden hamsters causes a high incidence of diverse neoplasms, all characterized by the SV40-specific, nuclear "T"-antigen (Diamandopoulos 1972, 1973). Diamandopoulos et al (1976) have recently shown that SV40-induced, neoplastic lymphoid cells, e. g. GD248 cells are phenotypically stable during repeated -_I in vitro subculture. However, we noticed that explants of GD248 tumors always yield a small proportion of SV40 "'I''-antigen-positive adherent cells with phenotypes distinct from that of parental GD248 lymphocytes. We have now characterized several lines of such adherent cells and are led to conclude that they may originate from --in __viva fusion of GD248 lymphocytes with host cells. In each of a series of four attempts to establish cells from GD248 tumors in vitro, we discovered that while most of the cells grew in suspension, a small proportion propagated adherent to the walls of the culture flasks. In two cases, these cells were estaolished in independent culture. For this the suspended cells were removed, the medium replaced and culture continued. After lo-20 days the adherent cells attained confluence and were then subcultured to give two lines, T16 and T17. T16 cells were propagated on a scale large enough for transfer to hamsters, where they developed tumors. The cells of these tumors were then again transferred to
12
Cell
Biology
International
Reports,
Vol.
2, No.
1, 19 78
culture, yielding only adherent cells. Two additional cell lines, T18 and T19 were isolated this way. Suspension cells and adherent cells were compared by light microscopy evaluating both living cells and cells fixed in methanol and colored with Giemsa stain. The suspension cells had the characteristics of lymphocytes. However, viable or fixed subconfluent adherent cells invariably exhibited polygonal and fusiform morphology In confluent cultures, cellular orientation was irregular with considerable nuclear overlap and numerous giant cells. Scanning electron microscopy of the GD248 cells revealed characteristics of peripheral-blood, splenic or lymph node lymphocytes with numerous, long microvilli (Fig. 1). The adherent cells from confluent cultures, exhibited few microvilli near the nuclear region, or none at all. Contact between cells occurred via thin cytoplasmic processes (Fig. We also observed rounded cells, with the surface topology of 2 1. cells in mitosis. Transmission electron microscopy showed the morphology of the suspension cells to be typical of lymphoid cells, contrasting with that of the adherent cells which exhibited extensive rough endoplasmic reticulum, many free polyribosomes, abundant occasional desmosomes and hemiGolgi apparatus, frequent lysosomes, desmosomes, a large nucleoli, surrounded by nucleolar-associated chromatin and microfibrils near the nuclear SV40 "T" antigen, as determined by the indirect immunofluorescent technique. Thirteen of eighteen 6-week old hamsters inoculated subcutaneously with 0.5 x 108 adherent cells developed 1 cm tumors within two weeks. The other five animals showed either no tumor or regression within four weeks. Six animals were tested for intraperitoneal tumor developTwo animals received 1 x 10' cells and showed no evidence of ment. tumor after three weeks. The four animals that received more than 1 x 10' cells exhibited extensive tumor growth within two weeks.The tumor cells were all SV40 "T''-antigen positive, indicating SV40 transformation. Moreover, sera obtained from the tumor-bearing animals contained anti-"T"-antibody. To characterize the mitotic adherent and GD248 lymphoid cells, we have determined the ploidy and karyotypes. Cells from GD248 tumors exhibited at least two hyperdiploid modal values, 46 and 48 GD248 cells (30% each), plus cells in the hypertetraploid range. The metacultured in suspension for 48 hrs. gave similar results. phase cells in the hyperdiploid range showed extensive karyotypic variation and numerous chromosomal aberrations. In contrast, the adherent cells consist primarily of hypertetraploid and pclyploid individuals (Table I).
Cell Biology
International
Table I
Reports,
Vol. 2, No. 7, 7 9 78
73
Ploidy of GD248 Tumor Cells and Adherent Isolated from GD248 Tumors -___ in vitro
Tumor or Cell Lines
Range of Chromosome Number 4Nt >lOO 2N*
GD248 tumor
185
T16a
Cells Total Cells Counted 227
42
14
194
605
813
T17
1
80
326
407
T18
2
47
157
206
GD248 (A)
185
16
6
207
GD248 (B)
89
10
3
101
T40b
11
120
78
204
a Cell lines T16, T17 and T18 were isolated from GD248 tumors. b Cell lines T44 and T40 were isolated from suspension cell line GD248 (A) and GD248 (B) induced tumors respectively. Two-dimensional immune electrophoresis (Schmidt-Ullrich et al, 1976) using rabbit anti-hamster-immunoglobulin (Grand Island Biological Co.) demonstrated membrane-bound immunoglobulin in GD248 lymphocytes but gave no detectable reactions for immunoglobulin for either the membrane of the adherent cells or their cytosol. Adherent cells actively ingest carbon black particles (Diaman-, dopoulos et al, 1976) but GD248 cells are non-phagocytic. The original GD248 tumors consist primarily of well-differentiated lymphocytes (Fig. 3), as described before (Diamandopoulos, 1973; Diamandopoulos, et al, 1976), with few poorly-differentiated cells. In contrast, adherent cells produce tumors (Fig. 4) composed of very poorly differentiated cells, frequent giant cells and cells in mitosis, all with large nuclei and cytoplasm. GD248 lymphoid cells carried in vitro, e. g. lines CD248 A and GD248 B (Diamandopoulos et al, 1976) have stable phenotypes. It is therefore unlikely that our adherent cells are directly derived from GD248 lymphocytes. Also, GD248 cells release no infectious virus (Diamandopoulos 1972, 1973) and it is improbable that the adherent cells are -in vitro transformants of normal cells contaminating the GD248 explants. However, it is possible that the adherent cells represent trace SV40-induced components of GD248 tumors or that they are hybrids of GD248 lymphocytes and host cells such as have been demonstrated for other --in vivo tumor systems
Cell Biology
International
Reports,
Vol. 2, No.
7, 1978
Fig. 1 (Upper left) Scanning elect ron micrograph of suspension culmicrograph tured GD248 cells. Fig. 2 (Upper r ,ight) Scanning electron Light of adherent cells in monolayer cul ture. Fig. 3 (Lower left) Fig. 4 micrograph of a hamster GD248 tumor (Hematoxylin/eosin). (Lower right) Light micrograph of a tumor removed from a hamster (Hematoxyln/eosin, magnification after the injection of T16 cells. same as Fig. 3.
Cell Biology
lnternationaf
Reports,
Vol. 2, No. I, 1978
Fig. 5 (Upper left) Phase contrast micrograph of a primary culture of cells from GD248 (b) cells induced tumor. Fig. 6 (Upper right) Phase contrast micrograph of subcultured adherent cells as in Fig.5 (same magnification as in Fig. 5). Fig. 3 (Lower left) SV40 "Tcantigen positive cells with various shaped nucleus from a primary cul.ture of tumor induced by GD248 (B) cells. Fig. 8 (Lower right) Light micrograph of a tumor removed from a hamster after the injection of adherent cells isolated from GD248 (A) cells induced tumor.
16
Cell Biology
International
Reports,
Vol. 2, No.
1, 1978
(Fenyo et al, 1973; Goldenberg et al, 1974; Wiener, et al, 1972, 1974). TO distinguish between the last two possibilities, cells from GD248 A and GD248 B (Diamandopoulos et al, 1976), were transplanted into hamsters. Tumor cells derived from these transplants were used to test for the presence of adherent cells (Figs. 5, 6) and for further 3 in vivo passages, both --in vivo passages. After --GD248 A and GD248 B tumors yielded small proportions of adherent, SV40 "T"-antigen positive (Fig. 7) polyploid (Table I), phagocytic cells. Two lines, T44 from GD248 A and T40 from GD248 B, were studied further (Table I). Both produced tumors with a histology (Fig. 8) different from that of the original GD248 tumors and resembling reticulum cell sarcoma. We suspect that the adherent cells have arisen from --in vivo somatic fusion of GD248 lymphoid cells and phagocytic host cells. This histology of the tumors showing many phagocytic cells intermixed with the lymphoid cells supports this possibility. METHODS GD248 tumors were removed from anesthetized animals, lightly minced to obtain individual cells and washed in Hanks' balanced salt solution (GIBCO, Grand Island, New York). Primary cultures were set up in plastic flasks (Falcon, Cockeyville, Md) at a density of 1.5 x lo7 Trypan blue-negative cells per ml of RPM1 1640 (ABS co., Buffalo, fetal calf New York), 10% in heat-inactivated serum (GIBCO) and then incubated at 37°C in a humidified 5% CO2 95% air. GD248 cell lines (GD248 A and GD248 B) were obtained from Dr. G. Diamandopoulos and propagated in suspension as in Diamandopoulos et al (1976). Scanning electron microscopy was as in Lin et For transmission electron microscopy, suspended cells al, (1973). and adherent cells removed with a rubber policeman, were pelleted followed by 1% osmium tetroxide, and fixed with 2% glutaraldehyde, and embedding in Epon (Fisher Scigraded dehydration with ethanol, entific Co., Fair Lawn, New Jersey). Ultrathin sections, cut on a Mt-1 microtome, were stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed with a Philips 300 electron microscope operated at 60 kV. SV40 "T"-antigen in adherent cells grown on coverslips or suspended GD248 cells smeared on coverslips was determined by the method of Rapp et al (1964). To determine the tumorigenicity of the adherent cells, these were scraped off, washed twice with 0.15 M NaCl, 0.05 M phosphate, pH 7.2, and injected intraperitoneally or subcutaneously into 6-week old outbred Syrian golden hamsters. The doses were 7 x lo7 to 1 x lo8 cells in 0.5 to 2ml. For tumor histology, tumor fragments were fixed in buffered neutral formalin (10%) embedded in paraffin and 5 pm sections To determine ploidy and karyostained with hematoxylin/eosin. types, cells from cultures and tumors were exposed to colcemid (final concentration 0.05 ug/ml) for 2 hr at 37"C, transferred to hypotonic (0.075 M) KC1 and, after 10 min, fixed in glacial acetic/ methanol (l/3), deposited on microscope slides and colored with Cells in mitosis were evaluated according to IshiGiemsa stain. hara et al (1962).
Ce// Biology
International
Reports,
Vol.
2, No.
I, 1978
17
ACKNOWLEDGEMENTS We thank Dr. R. Schmidt-Ullrich for the immunoelectrophoreses, Drs. H.J. Wolfe and P. Lui for histological preparation, Dr. R.A. DeLellis for discussion and suggestions and Dr. G. Th. Diamandopoulos and Dr. M.H. Miller for providing suspension cultured cell lines GD 248 A and GD 248 B. These studies were supported by a contract (CB-44000) from the National Cancer Institute and by Grant CA-12178 from the U.S. Public Health Service. REFERENCES Diamandopoulos, G.Th. (1972) Leukemia, lymphoma and osteosarcomainduced in the Syrian golden hamster by Simian Virus 40. Science 176, 173-175. Diamandopoulos, G.Th. (1973) Induction of lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma and osteogenic sarcoma in the Syrian golden hamster by oncogenic DNA Simian Virus 40. Journal of the National Cancer Institute 50, 1347-1365. Diamandopoulos, G.Th., Miller, M.H., McLane, M.F. and Evans, P. G. (1976) Loss or persistence of the differentiated state of Simian Virus 40-induced hamster tumor cells before and after serial passage in culture. Cancer Research 36, 3171-3177. Feny;, E.M., Wiener, F., Klein, G. and Harris, H. (1973) Selection of tumor-host cell hybrids from polyoma virusand methylcholanthrene-induced sarcoma. Journal of the National Cancer Institute 51, 1865-1875. Goldenberg, D.M., Pavia, R.A. and Tsao, M.L. (1974) In vivo hybridization of human tumor and normal hamster cells. Nature 250, 639-641. Tshihara, T., Moore, G.E. and Sandberg, A.A. (1962) Chromosome constitution of tumors of the golden hamster. Journal of the National Cancer Institute 29, 161-195. Lin, P.S., Wallach, D.F.H. and Tsai, S. (1973) Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proceedings cf the National Academy of Sciences U.S.A. 70, 2492-2496. J.S. and Melnick, J.L. (1964) Virus-induced intraRaw, F., Butel, nuclear antigen in cells transformed by papovavirus SV 40. Proceedings of the Society of Experimental Biology and Medicine 116, 1131-1135. Reynolds, Et.S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microsocpy. Journal of Cell Biology 17, 208-212. Schmidt-Ullrich, R., Wallach, D.F.H. ant Davis II, F.D.G. (1976) Membranes of normal hamster lymphocytes and lymphoid. cells neoplastically transformed by Simian Virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional immune electrophoresis, Journal of the National Cancer Institute 57, 1117-1126.
18
Cell Biology
International
Reports,
Vol. 2, No. I, 19 78
Wiener, F., Fenyg, E.M., Klein, G. and Harris, H. (1972) Fusion of Nature New Biology 238, 155-157. tumor cells wi;h host cells. Wiener, F., Fenyo, E.M. and Klein, G. (1974) Tumor-host cell hybrid in radiochimeras. Proceedings of the National Academy of Sciences 71, 148-152.
International
Phenotypic
Reports,
modification lymphoid
Vol. 2, No. 1, 1978
of SV40-transformed cells -in vivo
hamster
Peck-Sun Lin*, Catherine E. Butterfield, and Donald F. H. Wallach Tufts-New England Medical Center Therapeutic Radiology Department Radiobiology Division 171 Harrison Avenue Boston, Mass. 02111 * To whom correspondenc:e
should be addressed
ABSTRACT Explants of simian virus 40 (SV40)-induced lymphoid tumors yield SV40-T-antigen-positive derivatives that differ from GD248 lymphocytes propagated in suspension culture, (or -in vivo), in the substrates following respects: polygonal shape, adhesion to culture phagocytic capacity, lack of immunoglobulin and a chromo--in vitro, When GDsome complement at least twice that of GD248 lymphocytes. 248 lymphocytes are propagated as suspension in vitro, no such adherent variants can be detected. However, sequential in vivo pas-__ sage of GD248 lymphocytes obtained Erom the suspension-culture lines also yield adherent cell lines upon explantation in vitro-In-jection of adherent cells into hamsters produces tumors with histological features of reticulum cell ,sarcoma. Injection of Simian Virus 40 (SV40) into three-week old outbred Syrian golden hamsters causes a high incidence of diverse neoplasms, all characterized by the SV40-specific, nuclear "T"-antigen (Diamandopoulos 1972, 1973). Diamandopoulos et al (1976) have recently shown that SV40-induced, neoplastic lymphoid cells, e. g. GD248 cells are phenotypically stable during repeated -_I in vitro subculture. However, we noticed that explants of GD248 tumors always yield a small proportion of SV40 "'I''-antigen-positive adherent cells with phenotypes distinct from that of parental GD248 lymphocytes. We have now characterized several lines of such adherent cells and are led to conclude that they may originate from --in __viva fusion of GD248 lymphocytes with host cells. In each of a series of four attempts to establish cells from GD248 tumors in vitro, we discovered that while most of the cells grew in suspension, a small proportion propagated adherent to the walls of the culture flasks. In two cases, these cells were estaolished in independent culture. For this the suspended cells were removed, the medium replaced and culture continued. After lo-20 days the adherent cells attained confluence and were then subcultured to give two lines, T16 and T17. T16 cells were propagated on a scale large enough for transfer to hamsters, where they developed tumors. The cells of these tumors were then again transferred to
12
Cell
Biology
International
Reports,
Vol.
2, No.
1, 19 78
culture, yielding only adherent cells. Two additional cell lines, T18 and T19 were isolated this way. Suspension cells and adherent cells were compared by light microscopy evaluating both living cells and cells fixed in methanol and colored with Giemsa stain. The suspension cells had the characteristics of lymphocytes. However, viable or fixed subconfluent adherent cells invariably exhibited polygonal and fusiform morphology In confluent cultures, cellular orientation was irregular with considerable nuclear overlap and numerous giant cells. Scanning electron microscopy of the GD248 cells revealed characteristics of peripheral-blood, splenic or lymph node lymphocytes with numerous, long microvilli (Fig. 1). The adherent cells from confluent cultures, exhibited few microvilli near the nuclear region, or none at all. Contact between cells occurred via thin cytoplasmic processes (Fig. We also observed rounded cells, with the surface topology of 2 1. cells in mitosis. Transmission electron microscopy showed the morphology of the suspension cells to be typical of lymphoid cells, contrasting with that of the adherent cells which exhibited extensive rough endoplasmic reticulum, many free polyribosomes, abundant occasional desmosomes and hemiGolgi apparatus, frequent lysosomes, desmosomes, a large nucleoli, surrounded by nucleolar-associated chromatin and microfibrils near the nuclear SV40 "T" antigen, as determined by the indirect immunofluorescent technique. Thirteen of eighteen 6-week old hamsters inoculated subcutaneously with 0.5 x 108 adherent cells developed 1 cm tumors within two weeks. The other five animals showed either no tumor or regression within four weeks. Six animals were tested for intraperitoneal tumor developTwo animals received 1 x 10' cells and showed no evidence of ment. tumor after three weeks. The four animals that received more than 1 x 10' cells exhibited extensive tumor growth within two weeks.The tumor cells were all SV40 "T''-antigen positive, indicating SV40 transformation. Moreover, sera obtained from the tumor-bearing animals contained anti-"T"-antibody. To characterize the mitotic adherent and GD248 lymphoid cells, we have determined the ploidy and karyotypes. Cells from GD248 tumors exhibited at least two hyperdiploid modal values, 46 and 48 GD248 cells (30% each), plus cells in the hypertetraploid range. The metacultured in suspension for 48 hrs. gave similar results. phase cells in the hyperdiploid range showed extensive karyotypic variation and numerous chromosomal aberrations. In contrast, the adherent cells consist primarily of hypertetraploid and pclyploid individuals (Table I).
Cell Biology
International
Table I
Reports,
Vol. 2, No. 7, 7 9 78
73
Ploidy of GD248 Tumor Cells and Adherent Isolated from GD248 Tumors -___ in vitro
Tumor or Cell Lines
Range of Chromosome Number 4Nt >lOO 2N*
GD248 tumor
185
T16a
Cells Total Cells Counted 227
42
14
194
605
813
T17
1
80
326
407
T18
2
47
157
206
GD248 (A)
185
16
6
207
GD248 (B)
89
10
3
101
T40b
11
120
78
204
a Cell lines T16, T17 and T18 were isolated from GD248 tumors. b Cell lines T44 and T40 were isolated from suspension cell line GD248 (A) and GD248 (B) induced tumors respectively. Two-dimensional immune electrophoresis (Schmidt-Ullrich et al, 1976) using rabbit anti-hamster-immunoglobulin (Grand Island Biological Co.) demonstrated membrane-bound immunoglobulin in GD248 lymphocytes but gave no detectable reactions for immunoglobulin for either the membrane of the adherent cells or their cytosol. Adherent cells actively ingest carbon black particles (Diaman-, dopoulos et al, 1976) but GD248 cells are non-phagocytic. The original GD248 tumors consist primarily of well-differentiated lymphocytes (Fig. 3), as described before (Diamandopoulos, 1973; Diamandopoulos, et al, 1976), with few poorly-differentiated cells. In contrast, adherent cells produce tumors (Fig. 4) composed of very poorly differentiated cells, frequent giant cells and cells in mitosis, all with large nuclei and cytoplasm. GD248 lymphoid cells carried in vitro, e. g. lines CD248 A and GD248 B (Diamandopoulos et al, 1976) have stable phenotypes. It is therefore unlikely that our adherent cells are directly derived from GD248 lymphocytes. Also, GD248 cells release no infectious virus (Diamandopoulos 1972, 1973) and it is improbable that the adherent cells are -in vitro transformants of normal cells contaminating the GD248 explants. However, it is possible that the adherent cells represent trace SV40-induced components of GD248 tumors or that they are hybrids of GD248 lymphocytes and host cells such as have been demonstrated for other --in vivo tumor systems
Cell Biology
International
Reports,
Vol. 2, No.
7, 1978
Fig. 1 (Upper left) Scanning elect ron micrograph of suspension culmicrograph tured GD248 cells. Fig. 2 (Upper r ,ight) Scanning electron Light of adherent cells in monolayer cul ture. Fig. 3 (Lower left) Fig. 4 micrograph of a hamster GD248 tumor (Hematoxylin/eosin). (Lower right) Light micrograph of a tumor removed from a hamster (Hematoxyln/eosin, magnification after the injection of T16 cells. same as Fig. 3.
Cell Biology
lnternationaf
Reports,
Vol. 2, No. I, 1978
Fig. 5 (Upper left) Phase contrast micrograph of a primary culture of cells from GD248 (b) cells induced tumor. Fig. 6 (Upper right) Phase contrast micrograph of subcultured adherent cells as in Fig.5 (same magnification as in Fig. 5). Fig. 3 (Lower left) SV40 "Tcantigen positive cells with various shaped nucleus from a primary cul.ture of tumor induced by GD248 (B) cells. Fig. 8 (Lower right) Light micrograph of a tumor removed from a hamster after the injection of adherent cells isolated from GD248 (A) cells induced tumor.
16
Cell Biology
International
Reports,
Vol. 2, No.
1, 1978
(Fenyo et al, 1973; Goldenberg et al, 1974; Wiener, et al, 1972, 1974). TO distinguish between the last two possibilities, cells from GD248 A and GD248 B (Diamandopoulos et al, 1976), were transplanted into hamsters. Tumor cells derived from these transplants were used to test for the presence of adherent cells (Figs. 5, 6) and for further 3 in vivo passages, both --in vivo passages. After --GD248 A and GD248 B tumors yielded small proportions of adherent, SV40 "T"-antigen positive (Fig. 7) polyploid (Table I), phagocytic cells. Two lines, T44 from GD248 A and T40 from GD248 B, were studied further (Table I). Both produced tumors with a histology (Fig. 8) different from that of the original GD248 tumors and resembling reticulum cell sarcoma. We suspect that the adherent cells have arisen from --in vivo somatic fusion of GD248 lymphoid cells and phagocytic host cells. This histology of the tumors showing many phagocytic cells intermixed with the lymphoid cells supports this possibility. METHODS GD248 tumors were removed from anesthetized animals, lightly minced to obtain individual cells and washed in Hanks' balanced salt solution (GIBCO, Grand Island, New York). Primary cultures were set up in plastic flasks (Falcon, Cockeyville, Md) at a density of 1.5 x lo7 Trypan blue-negative cells per ml of RPM1 1640 (ABS co., Buffalo, fetal calf New York), 10% in heat-inactivated serum (GIBCO) and then incubated at 37°C in a humidified 5% CO2 95% air. GD248 cell lines (GD248 A and GD248 B) were obtained from Dr. G. Diamandopoulos and propagated in suspension as in Diamandopoulos et al (1976). Scanning electron microscopy was as in Lin et For transmission electron microscopy, suspended cells al, (1973). and adherent cells removed with a rubber policeman, were pelleted followed by 1% osmium tetroxide, and fixed with 2% glutaraldehyde, and embedding in Epon (Fisher Scigraded dehydration with ethanol, entific Co., Fair Lawn, New Jersey). Ultrathin sections, cut on a Mt-1 microtome, were stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed with a Philips 300 electron microscope operated at 60 kV. SV40 "T"-antigen in adherent cells grown on coverslips or suspended GD248 cells smeared on coverslips was determined by the method of Rapp et al (1964). To determine the tumorigenicity of the adherent cells, these were scraped off, washed twice with 0.15 M NaCl, 0.05 M phosphate, pH 7.2, and injected intraperitoneally or subcutaneously into 6-week old outbred Syrian golden hamsters. The doses were 7 x lo7 to 1 x lo8 cells in 0.5 to 2ml. For tumor histology, tumor fragments were fixed in buffered neutral formalin (10%) embedded in paraffin and 5 pm sections To determine ploidy and karyostained with hematoxylin/eosin. types, cells from cultures and tumors were exposed to colcemid (final concentration 0.05 ug/ml) for 2 hr at 37"C, transferred to hypotonic (0.075 M) KC1 and, after 10 min, fixed in glacial acetic/ methanol (l/3), deposited on microscope slides and colored with Cells in mitosis were evaluated according to IshiGiemsa stain. hara et al (1962).
Ce// Biology
International
Reports,
Vol.
2, No.
I, 1978
17
ACKNOWLEDGEMENTS We thank Dr. R. Schmidt-Ullrich for the immunoelectrophoreses, Drs. H.J. Wolfe and P. Lui for histological preparation, Dr. R.A. DeLellis for discussion and suggestions and Dr. G. Th. Diamandopoulos and Dr. M.H. Miller for providing suspension cultured cell lines GD 248 A and GD 248 B. These studies were supported by a contract (CB-44000) from the National Cancer Institute and by Grant CA-12178 from the U.S. Public Health Service. REFERENCES Diamandopoulos, G.Th. (1972) Leukemia, lymphoma and osteosarcomainduced in the Syrian golden hamster by Simian Virus 40. Science 176, 173-175. Diamandopoulos, G.Th. (1973) Induction of lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma and osteogenic sarcoma in the Syrian golden hamster by oncogenic DNA Simian Virus 40. Journal of the National Cancer Institute 50, 1347-1365. Diamandopoulos, G.Th., Miller, M.H., McLane, M.F. and Evans, P. G. (1976) Loss or persistence of the differentiated state of Simian Virus 40-induced hamster tumor cells before and after serial passage in culture. Cancer Research 36, 3171-3177. Feny;, E.M., Wiener, F., Klein, G. and Harris, H. (1973) Selection of tumor-host cell hybrids from polyoma virusand methylcholanthrene-induced sarcoma. Journal of the National Cancer Institute 51, 1865-1875. Goldenberg, D.M., Pavia, R.A. and Tsao, M.L. (1974) In vivo hybridization of human tumor and normal hamster cells. Nature 250, 639-641. Tshihara, T., Moore, G.E. and Sandberg, A.A. (1962) Chromosome constitution of tumors of the golden hamster. Journal of the National Cancer Institute 29, 161-195. Lin, P.S., Wallach, D.F.H. and Tsai, S. (1973) Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proceedings cf the National Academy of Sciences U.S.A. 70, 2492-2496. J.S. and Melnick, J.L. (1964) Virus-induced intraRaw, F., Butel, nuclear antigen in cells transformed by papovavirus SV 40. Proceedings of the Society of Experimental Biology and Medicine 116, 1131-1135. Reynolds, Et.S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microsocpy. Journal of Cell Biology 17, 208-212. Schmidt-Ullrich, R., Wallach, D.F.H. ant Davis II, F.D.G. (1976) Membranes of normal hamster lymphocytes and lymphoid. cells neoplastically transformed by Simian Virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional immune electrophoresis, Journal of the National Cancer Institute 57, 1117-1126.
18
Cell Biology
International
Reports,
Vol. 2, No. I, 19 78
Wiener, F., Fenyg, E.M., Klein, G. and Harris, H. (1972) Fusion of Nature New Biology 238, 155-157. tumor cells wi;h host cells. Wiener, F., Fenyo, E.M. and Klein, G. (1974) Tumor-host cell hybrid in radiochimeras. Proceedings of the National Academy of Sciences 71, 148-152.
