Tumor-Specific Antigens on Rat Liver Cells Transformed In Vitro by Chemical Carcinogens 1 T. Yokota, 2,3 P. Sizaret, 4 and N. Martel 4 , 5
ABSTRACT-With the use of membrane immunofluorescence and xenogeneic antisera, tumor-specific membrane antigens were detected on rat eplthelial-lIke liver cells transformed in vltro by chemlcal carclnogens. These antigens were not detected In 10-, 15-, and 19-day rat fetuses. Xenogenelc antlsera were produced In rabblts by immunlzatlon of the rabblts wlth cultlvated SO rat Iiver cells transformed by dimethylnitrosamine or Nmethyl-N'-nltro-N-nltrosoguanldlne. The speclflc antlsera agalnst tumor-assoclated antlgen(s) were obtained by In vlvo absorption In syngenelc male rats and by In vltro absorption wlth varlous cell IInes. One tumor-speclflc Individual antigen and two tumorspeclflc cross-reactlng antigens were shown to be present on the surface of chemlcally and/or spontaneously transformed rat IIver cell lines. They were not detected on liver and spleen cells of normal SO adult rats, on fetal liver cells, or on Iiver and Intestinal carclnoma cells of Wistar rats. Sera from multlparous pregnant rats had no antlbodles agalnst these tumor antigens (although they reacted with fetal cells).-J Natl Cancer Inst 60: 125-129, 1978.
Various tumor-specific or tumor-associated antigens are expressed on the surfaces of malignant cells derived from chemically induced tumors (l, 2). Similar antigens can also be expressed on cells transformed in vitro. For example, tumor-specific individual or cross-reacting antigens have been detected on mouse prostate cells (3, 4) and rat liver cells (5) transformed in vitro. However, we do not know whether oncofetal antigens are expressed on cells transformed in vitro, although such antigens have been detected on cells derived from tumors induced with chemical carcinogens (6, 7). Using the membrane immunofluorescence technique with xenogeneic antisera, Martin et al. (8) recently detected a tissue-specific oncofetal antigen on rat cells originating from a chemically induced intestinal carcinoma. In the present study, oncofetal antigens on rat epithelial-like liver cell lines transformed in vitro were sought by indirect immunofluorescence with sera from xenogeneic hosts and from multiparous pregnant rats. Such oncofetal antigens were not detected; however, one individual and two cross-reacting tumor-specific antigens were found.
ABBREVIATIONS USED: DMN = dimethylnitrosamine; MNNG methyl-N' -nitro-N-nitrosoguanidine.
=
N-
Received February 4,1977; accepted August 3,1977. The work reported in this paper was undertaken during the tenure of a Corvissiano Fellowship awarded by the International Agency for Research on Cancer, Lyon, France. 3 Department of Bacteriology, Fukushima Medical College, Fukushima, 960 Japan. 4 International Agency for Research on Cancer (IARC), 150 cours Albert Thomas, 69008 Lyon, France. 5 We are indebted to the following persons: Professor F. Martin , University of Dijon, Dijon , France, for supplying the DHD BD IX intestinal carcinoma cellline; Dr. M. Aupoix, Institut National de la Sante et de la Recherche Medicale , Lyon, for supplying the 3T3-FL Gross virus-infected cell line; Professor R. Baldwin, the Cancer Research Campaign Laboratories, University of Nottingham, Nottingham, England, for supplying the D 202 Wistar he patorna cell line; Dr , P. Chany, Institut de Recherehes Scientifiques sur le Cancer, Villejuif, France, for supplying the LF Wistar hepatoma cell line; Professor P. Padieu, University of Dijon, for supplying the 18 PC-FR Wistar fetalliver cellline; Dr. J. Desgres, Faculte Mixte de Merleeine et de Pharmacie, Dijon, for controlling the metabolis m of progesterone into 6-alpha-hydroxypregnenolone by cell lines of liver origin; Dr. C. Samarut, Höpital Edouard Herriot, Lyon, for doing the erythrocyte-antibody rosette assay; and Professor H. Hirai and Dr. S. Nishi, both of the Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan, for providing the test system for rat (lfetoprotein. We thank Mrs. L. Saint- Vincent and Miss C. Drevon, both of the IARC, for technical help; Dr. T. Kuroki, Dr. J. P. Lamelin, and Dr. G. Lenoir, all of the IARC, and Professor J. P. Revillard, Höpital Edouard Herriot, for useful discussions; Dr. C. A. LinselI, IARC, for his constant support; and Miss S. Reynaud, IARC, for typing this manuscript. 1
2
MATERIALS AND METHODS Animals.-BD IV and BD VI rats were used. They have been maintained inbred since 1970 by systematic brother X sister matings at the International Agency for Research on Cancer in Lyon. Cell cultures.- The rat cell lines and tissue material used are listed in tables 1 and 2. They included epitheVOL. 60, NO. 1, JANUARY 1978
lial-like cell lines IAR 2 and IAR 20 derived from livers of 10-day-old BD VI rats and IAR 6 derived from the liver of an 8-week-old BD IV rat, as weIl as corresponding malignant cell lines obtained either by spontaneous malignant transformation or after in vitro treatment with DMN or MNNG (9-11). IAR 6-1 RT-7 was derived from a tumor induced in a BD IV rat by ip injection of the IAR 6-1 cell line. We verified that the malignant liver cell lines originated in the liver by noting the appearance of a rat liver-specific membrane antigen (12) and by controlling the capacity of the cell lines to metabolize the progesterone in 6-alpha-hydroxy-pregnenolone (13, 14). The malignancy of transformed cell lines was verified by their sc or ip injection into syngeneic newborn rats (10). Cell lines were maintained in tissue culture flasks (Falcon Plastics, Oxnard, Calif.) with Williams-D medium supplemented with 10% heatinactivated fetal bovine serum (Flow Laboratories, Rockville, Md.). They were serially passaged after 0.25% trypsin dissociation.
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Xenogeneic antisera.-Rabbits were immunized with cultured living cells suspended by trypsinization. They received three to five ip injections of 1-3 x 107 cells in 2 ml phosphate-buffered saline every 2 weeks and were bled 10 days after the last injection. The antibodies directed against the normal antigenic components of rats were eliminated by in vivo absorption (15). Syngeneic male rats were given ip injections of 0.1-10 ml rabbit antiserum per kilogram body weight and were bled by cardiac puncture 16 hours later. Negative controls were given injections of normal rabbit serum. To determine whether the positive immunofluorescence reactions corresponded to individual or cross-reacting antigens, additional absorptions were done in vitro as follows: Living cells were suspended at a final concentration of 5x 107/ml of serum previously absorbed in vivo. After incubation for 2 hours at 37° C, the cells were removed by centrifugation. The in vitro absorptions were each performed three times . Sera from pregnant rats.-Blood was taken at day 1520 of gestation or 7 days after the last delivery from pregnant BD rats having had at least 3 litters. Negative control sera were obtained from virgin female rats. Membrane immunojluorescence tests.-Immune sera were assayed against target cells by indirect membrane immunofluorescence, as described by Martin et al. (8). Fluorescein isothiocyanate conjugates had the following origin: Anti-rabbit IgG sheep serum came from the Institut Pasteur (Paris, France), and anti-rat IgG goat serum was purchased from H yland Laboratories (Los Angeles, Calif.). Membrane immunofluorescence was
examined with a Tiyoda fluorescence microscope. Fluorescence indexes were calculated as the percentage of cells unstained by negative control serum minus the percentage of cells unstained by the test sera divided by the percentage of cells unstained by negative control serum (16). The percentages of cells unstained by negative control serum and by fluorescence reagent alone were over 99.5%. RESULTS Reaction 01 Xenogeneic Antisera Absorbed In Vivo
The reactions of xenogeneic antisera after in vivo absorption are indicated in table 1. Of 7 rabbit antisera, 3 reacted with the corresponding immunizing celllines (IAR 6-1, IAR 2-31, and IAR 20 PCI-3). The reactivity of anti-IAR 6-1 was restricted to the isologous cell line and to the derived IAR 6-1 RT-7 cell line. However, anti-IAR 2-31, in addition to reacting with the immunizing cell line, reacted with several malignant cell lines derived from IAR 2 and 6, transformed either chemically or spontaneously. Similarly, anti-IAR 20 PCI-3 reacted not only with the isologous cell line but also with malignant cell Iines derived from IAR 2; it did not react with the corresponding nonmalignant cell lines. None of the antisera reacted with normal rat cells derived from various organs, with cells taken from 10-, 15-, and 19-day fetuses (table 2), with intestinal carcinoma cells of BD IX rats, or with liver tumor cell Iines from Wistar rats (table 1).
TADLE l.-Membrane immunofluorescence ofvarious rat cells incubated with in vivo absorbed sera ofrabbits immunized against rat liver cells chemically transformed in vitro Fluorescence indexes" with antisera to: Original rat strain
BDIV BDIV BDVI BDVI BDIV BDIV BDIV BDVI BDVI BDVI BDVI BDIX Wistar Wistar BDIV BDVI Wistar a b C
d
e f
J
Target cells a Non transformed IAR6 IAR 6-10 IAR2 IAR20 Chemically transformed IAR 6-1 IAR 6-1 RT-7 d IAR 6-4 IAR 2-22 IAR 2-28 IAR 2-31 IAR 20 PCl-3 e DHD intestinal carcinoma/ LF hepatoma f D 202 hepatoma/ Spontaneously transformed IAR 6-7 IAR 2-25 18 PC-FR
Treatment of cell by"
IAR 6-1
IAR 6-1 IAR 6-4 RT-7
IAR 222
IAR 228
IAR 231
IAR20 PCl-3
Nothing MNNG Nothing Nothing
0.02 0.00 0.00 0.02
0.00 0.00 NT NT
NT 0.00 NT NT
0.00 NT 0.00 0.00
NT NT 0.00 0.00
0.07 0.00 0.03 0.00
0,00 0.00 0.12 0.09
DMN DMN DMN DMN MNNG MNNG MNNG DMH DAB DAB
0.97 0.92 0.00 0.00 0.02 0.00 0.01 0.00 0.00 0.00
0.00 0.05 0.00 NT NT 0.00 0.00 NT NT NT
0.00 0.00 0.00 NT NT 0.00 0.00 NT NT NT
0.00 NT 0.00 0.02 0.00 0.00 0.00 NT NT NT
0.00 0.00 NT NT 0.02 0.00 0.00 NT NT NT
0.55 0.82 0.85 0.85 0.82 0.93 0.12 0.00 0.00 0.00
0.00 0.00 0.19 0.88 0.82 0.79 0.88 0.00 0.00 0.00
Nothing Nothing Nothing
0.06 0.00 0.00
0.00 NT NT
0.00 NT NT
0.00 0.00 NT
0.00 0.00 NT
0.55 0.86 0.05
0.00 0.82 0.02
All celllines were ofliver origin except the intestinal carcinoma cellline that was derived from a tumor induced by DMH. DMH = 1,2-dimethylhydrazine; DAB =p-dimethylaminoazobenzene. A fluorescence index ofO.30 was considered positive. NT=not tested. Celllines derived from a tumor induced in a BD IV rat given an ip injection oflAR 6-1. Celllines derived from MNNG treatment oflAR 20PC, a cloning cellline from IAR 20. Celllines established from tumors.
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TUMaR-SPECIFIC ANTIGENS ON RAT LIVER CELLS TRANSFaRMED IN VITRa
TABLE 2.-Membrane immunofluorescence staining o{ cells {rom [reshly dissociated rat tissues incubated with in vivo absorbed sera o{ rabbits immunized against rat lioer cells chemically trans{ormed in »itro Fluorescence indexes with antisera to:" Rat strain
Age
Origin of target cells IAR 6-1
BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BDVI BDVI BDVI BDVI
10-day fetus 15-day fetus
Whole-body tissue Liver Whole-body tissue Liver Whole-body tissue Liver Spleen Intestine Lung Kidney
19-day fetus 12-wk-old rat
IAR 6-1 IAR 6-4 RT-7
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 NT 0.00 NT NT NT NT NT NT
IAR222
IAR228
IAR231
IAR 20 PCI-3
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
" N'I'r-not tested.
Reactions of Xenogeneic Antisera Absorbed In Vivo and In Vitro
Ta determine how many antigenic systems were present, the three antisera were absorbed further with isologaus and/or homologous cell lines (table 3). The TABLE 3.-Demonstration of tumor-specific individual and crossreacting antigens on malignant rat liuer cells by membrane immunofluorescence with rabbit antisera absorbed in vivo and in uitro Fluorescence indexes with:" Antisera to
In vitro absorption with"
IAR 6-4
IAR IAR 2-22 2-31
0.97 0.00 0.00 0.92
0.00 NT NT NT
0.00 NT NT NT
0.00 NT NT NT
0.01 NT NT NT
Nothing IAR 6-1 (DMN) IAR 2-22 (DMN) IAR 2-31 (MNNG) IAR 20 PCI-3 (MNNG) Fetal bovine serum
0.55 0.00 0.00 0.00 0.26 0.55
0.82 0.00 0.00 0.00 0.24 0.76
0.85 0.00 0.00 0.00 0.29 0.85
0.93 0.00 0.00 0.00 0.33 0.88
0.12 NT NT NT 0.00 NT
Nothing IAR 6-1 (DMN) IAR 2-31 (MNNG) IAR 20 PCI-3 (MNNG) Fetal bovine serum
0.00 0.00 0.00 0.00 NT
{Nothing IAR 6-1
IAR 2-31
IAR 20 PCI-3
IAR 20 PCI-3
IAR 6-1
IAR6-1 (DMN) IAR 6-1 RT-7" IAR 2-31 (MNNG)
0.19 0.00 0.00 0.00 NT
0.88 0.83 0.00 0.00 NT
0.79 0.75 0.00 0.00 NT
0.88 0.80 0.00 0.00 0.88
In vitro absorption was done three times with 5x lO' cells or 0.2 ml fetal bovine serum/ml of antiserum, b NT=not tested. c Cell Iine derived from a tumor induced in a BD IV rat given an injection of IAR 6-1.
31 could be eliminated with malignant ceillines divided from IAR 2 and 6; thus tumor-specific cross-reacting antigen(s) was expressed on these cell lines. Since the reactivity of anti-IAR 20 pe 1-3 was completely abolished by malignant cell lines derived from IAR 2 and 20, another tumor-specific cross-reacting antigen(s) was expressed on these ceillines.
Reactions of Sera From Multiparous Pregnant Rats Of the 7 assayed sera from multiparaus pregnant rats, 3 (#368, 555, and 395) reacted with freshly dissociated fetal cells from whole-body and liver tissues of 15-day-old BD IV arid/or BD VI rats with fluorescent indexes greater than 0.30 (table 4). However, they did not react with the 3 malignant IAR cell lines that carried either tumor-specific individual or cross-reacting antigens as evidenced by their reaction with heterologous sera.
DISCUSSION On the basis of other studies, we discarded the possibility that membrane immunofluorescence reacTABLE 4.-Membrane immunofluorescence o{cells [rom. uihole-body and lioer tissues o{ 15-day rat [etuses and [rom in oitro chemically transformed rat li ver cell lines after incubation with sera front multiparous pregnant rats Fluorescence indexes with:" Serum No.
No.of pregnancies
Rat strain
a
reactivity of anti-IAR 6-1 could be eliminated only with isologous cell lines IAR 6-1 and IAR 6-1 RT-7; therefore , only tumor-specific individual antigen(s) was expressed on these cell lines. The reactivity of anti-IAR 2-
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342 355 368 555 394 395 396
4 3 4 4
BD VI
5
BDIV
IAR cells chemically transformed in vitro
Fetal cells"
4 4
BD IV
BD VI
6-1
RT-7 2-31
NT NT 0.26 0.16 NT 0.30 0.12
0.13 0.00 0.33 0.40 0.08 0.48 0.17
NT NT 0.00 0.00 NT 0.00 0.00
NT NT 0.00 0.00 NT 0.00 0.00
NT NT 0.00 0.00 NT 0.00 0.00
20 PCI-3 NT NT 0.00 0.00 NT 0.00 0.00
" NT=not tested. b Freshly dissoeiated cells.
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YOKOTA, SIZARET, AND MARTEL
tions observed in this study were due to the binding through Fe receptors of nonspecific immune complexes present in absorbed rabbit antisera for the following reasons: a) Heat-aggregated immunoglobulins were not bound by the malignant cells (17), and b) malignant cells did not form erythrocyte-antibody rosettes when incubated with ox red blood cells coated with rabbit immunoglobulins (18). We concluded that the rat liver celilines transformed in vitro by chemical carcinogens acquired two different types of tumor-specific antigens: One type was absent from other malignant cell lines and was therefore considered to be an individual turnor-specific antigen; the other type was found on several malignant cell lines and was therefore considered to be tumor-specific crossreacting antigen. Oncofetal antigens have been detected by xenogeneic antisera on rat sarcoma cells (1) and on rat intestinal cells (8) from chemically induced tumors. Moreover, with the use of rat immune material such as lymphoid cells or sera from multiparous rats (6, 7), oncofetal antigens have been detected on a wide range of cells from chemically induced tumor. Techniques of assays included toxicity or membrane immunofluorescence. The scarcity of reports of oncofetal antigens on cell lines transformed in vitro (19) can be contrasted to the frequency of re ports of such antigens on cell lines derived from tumors. The present results are in line with the contrasting findings of those reports: Since the antigens we studied were not detectable on fetal cells taken at several stages of development and since none of the malignant cell lines reacted with sera from multiparous pregnant rats, these antigens seem to be different from oncofetal antigens. As expected, the nature of tumor-specific cross-reacting antigen(s) was not related to the ehernieals used for transformation; however, it seemed somehow related to the original cell line, as if only certain cell lines possess specific repressed potentialities that are realized only after malignant transformation. Moreover, because these antigens were not detected on malignant liver cell lines derived from Wistar rats transformed either in vitro or in vivo, they showed specificity to BD IV and BD VI rats. In this respect, these antigens differ from the PNantigen (chemically induced in vivo) detected by Okita et al. (20) on malignant hepatocytes and from the common antigen detected by Steele and Sjögren (21) on colon carcinoma cells. They appear to be of the same nature as the antigens, which were cross-reactive and which differed from the embryonie antigen, detected by Iype et al. (5) on rat liver cells transformed in vitro with chemical carcinogens. Since the techniques required for the study of tumorspecific transplantation antigens and tumor-specific surface antigens differ, we do not know whether individual tumor-specific surface antigens are identical to tumorspecific transplantation antigens. The observation in our study that only 1 of 6 malignant cell lines exhibited tumor-specific individual antigen can be explained by the use of xenogeneic antisera. Compared to homoloJ
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gous antisera, such antisera may be less likely to pick up small antigenic differences such as "individual malignant antigens." Another possible explanation is that , since most of our experiments were done on cell lines of polyclonal origin, individual antigenic determinants varied from cell to cell. The multiplicity of such determinants could result in antigenic competition. This may explain why tumor-specific cross-reacting antigens are more easily detected on malignant cell lines of polyclonal origin. Another possible reason that the antigens were not detected is the dilution of antisera during the in vivo absorption. It seems unlikely that the antigens detected in this study are induced by a sarcoma virus since, with the use of an antiserum against the gs-30 antigen of murine oncornavirus, no reaction was observed on any of the chemically transformed malignant cell lines and since none of our antisera reacted with the 3T3-FL celiline expressing virus antigens of murine oncornavirus after infection by the Gross virus. In addition, the supernatants of malignant cell lines did not induce the malignant transformation of any of the four original cell lines. As could be expected from the lack of reaction of rabbit anti-turnor-specific sera with rat fetal liver cells, the three antigens were found to be different from rat a-fetoprotein, since none of the malignant cell lines reacted by immunofluorescence with rabbit anti-rat afetoprotein. Further experiments will be necessary to ascertain whether the antigens reported have a role in the immune defense of the host.
REFERENCES (1) THoMsoN DM, ALEXANDER P: A cross-reacting embryonie anti-
(2)
(3)
(4) (5)
(6)
(7)
(8)
(9)
(10)
gen in the membrane of rat sarcoma cells which is immunogenic in the syngeneic host. Br] Cancer 27:35-47, 1973 ZOLLER M, PRICE MR, BALDWIN RW: Cell-mediated cytotoxicity to chemically-induced rat tumours. Int] Cancer 16:593-606, 1975 MONDAL S, IYPE PT, GRIESBACH L, et al: Antigens of cells derived from mouse prostate cell after malignant transforrnation in vitro by carcinogenic hydrocarbons. Cancer Res 30:1593-1600,1970 EMBLETON M], HEIDELBERGER C: Antigenicity of clones of mouse prostate cells transformed in vitro. Int] Cancer 9:8-18, 1972 IYPE PT, BALDWIN RW, GLAVES D: Cell surface antigenic changes induced in normal adult rat liver cells by carcinogen treatment in vitra. Br J Cancer 27:128-133,1973 BALDWIN RW, GLAVES D, VaSE BM: Ernbryonie antigen expression in chemically induced rat hepatomas and sarcomas. Int J Cancer 10:233-243, 1972 STEELE G], SJÖGREN HO: Embryonie antigens associated with chemically induced colon carcinomas in rat. Int J Cancer 14:435-442, 1974 MARTIN F, KNOBEL S, MARTIN M, et al: A carcinofetal antigen iocated on the membrane of cells from rat intestinal carcinoma in culture. Cancer Res 35:333-336, 1975 MONTESANO R, SAINT-VINCENT L, TOMATIS L: Malignant transformation in vitro of rat liver cells by dimethylnitrosamine and N-methyl-N'-nitro-N-nitrosoguanidine. Br J Cancer 28:215-220, 1973 MONTESANO R, SAINT-VINCENT L, DREVON C, et al: Production of epithelial and mesenchymal tumours with rat liver cells transformed in vitro. Int] Cancer 16:550-558,1976 VOL. 60, NO. 1, JANUARY 1978
TUMOR-SPECIFIC ANTIGENS ON RAT LIVER CELLS TRANSFORMED IN VITRO (11) KUROKI T, DREVON C, SAINT- VINCENT L, et al: Studies on the use of liver parenchymal cell in in vitro carcinogenesis. In Comptes-Rendus of the Centre National de Recherche Seientifique, Menton meeting, 1976. In press (12) !KAWA Y, NIWA A, TOMATIS L, et al: Transformation of a rat liver celliine by murine sarcoma virus. Proc Am Assoc Cancer Res 14:109, 1973 (13) DESGRES J, GUIGUET M, BEGUE RJ, et al: Etude du metabolisme he pato-foetal de la progesterone a l'aide de cellules hepatiques de rat en culture. In Comptes Rendus: Organisation des Laboratoires - Biologie Prospective: 3e Colloque de Pont-aMousson, 1975. Paris: Expansion Scientifique Francaise , 1975, pp 663-670 (14) - - - : Study of progesterone metabolism in foetal and postnatalliver cells in culture. In Advances in Mass Spectrometry, Biochemistry and Medicine (Frigerio A, Castagnoli N, eds). New York: Spectrum, 1976 (15) HERSEY P: The protective effect of antisera against leukaemia in
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vivo. A reappraisal. Br J Cancer 28:11-18,1973 (16) KLEIN E, KLEIN G: Antigenic properties of lymphomas induced by the Moloney agent. J Natl Cancer Inst 32:547-568, 1964 (17) DICKLER HB: Studies of the human lymphocyte receptor for he at aggregated or antigen-complexed immunoglobulin.J Exp Med 140:508-522, 1974 (18) BROCHIER J, SAMARUT C, REVILLARD JP: Rosette technique for identification of human mononuclear cells bearing Fe receptors. Biomedicine 23:206-209, 1975 (19) EMBLETON MJ, HEIDELBERGER C: Neoantigens on chemically transformed cloned C3H mouse embryo cells. Cancer Res 35:2049-2055, 1975 (20) OKITA K, KUGMAN LH, FARBER E: A new common marker for premalignant and malignant hepatocytes induced in the rat by chemical carcinogens. J Natl Cancer Inst 54: 199-201, 1975 (21) STEELE G JR, SJÖGREN HO: Cross-reacting tumor-associated antigen(s) among chemically induced rat colon carcinomas. Cancer Res 34:1801-1807,1974
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ABSTRACT-With the use of membrane immunofluorescence and xenogeneic antisera, tumor-specific membrane antigens were detected on rat eplthelial-lIke liver cells transformed in vltro by chemlcal carclnogens. These antigens were not detected In 10-, 15-, and 19-day rat fetuses. Xenogenelc antlsera were produced In rabblts by immunlzatlon of the rabblts wlth cultlvated SO rat Iiver cells transformed by dimethylnitrosamine or Nmethyl-N'-nltro-N-nltrosoguanldlne. The speclflc antlsera agalnst tumor-assoclated antlgen(s) were obtained by In vlvo absorption In syngenelc male rats and by In vltro absorption wlth varlous cell IInes. One tumor-speclflc Individual antigen and two tumorspeclflc cross-reactlng antigens were shown to be present on the surface of chemlcally and/or spontaneously transformed rat IIver cell lines. They were not detected on liver and spleen cells of normal SO adult rats, on fetal liver cells, or on Iiver and Intestinal carclnoma cells of Wistar rats. Sera from multlparous pregnant rats had no antlbodles agalnst these tumor antigens (although they reacted with fetal cells).-J Natl Cancer Inst 60: 125-129, 1978.
Various tumor-specific or tumor-associated antigens are expressed on the surfaces of malignant cells derived from chemically induced tumors (l, 2). Similar antigens can also be expressed on cells transformed in vitro. For example, tumor-specific individual or cross-reacting antigens have been detected on mouse prostate cells (3, 4) and rat liver cells (5) transformed in vitro. However, we do not know whether oncofetal antigens are expressed on cells transformed in vitro, although such antigens have been detected on cells derived from tumors induced with chemical carcinogens (6, 7). Using the membrane immunofluorescence technique with xenogeneic antisera, Martin et al. (8) recently detected a tissue-specific oncofetal antigen on rat cells originating from a chemically induced intestinal carcinoma. In the present study, oncofetal antigens on rat epithelial-like liver cell lines transformed in vitro were sought by indirect immunofluorescence with sera from xenogeneic hosts and from multiparous pregnant rats. Such oncofetal antigens were not detected; however, one individual and two cross-reacting tumor-specific antigens were found.
ABBREVIATIONS USED: DMN = dimethylnitrosamine; MNNG methyl-N' -nitro-N-nitrosoguanidine.
=
N-
Received February 4,1977; accepted August 3,1977. The work reported in this paper was undertaken during the tenure of a Corvissiano Fellowship awarded by the International Agency for Research on Cancer, Lyon, France. 3 Department of Bacteriology, Fukushima Medical College, Fukushima, 960 Japan. 4 International Agency for Research on Cancer (IARC), 150 cours Albert Thomas, 69008 Lyon, France. 5 We are indebted to the following persons: Professor F. Martin , University of Dijon, Dijon , France, for supplying the DHD BD IX intestinal carcinoma cellline; Dr. M. Aupoix, Institut National de la Sante et de la Recherche Medicale , Lyon, for supplying the 3T3-FL Gross virus-infected cell line; Professor R. Baldwin, the Cancer Research Campaign Laboratories, University of Nottingham, Nottingham, England, for supplying the D 202 Wistar he patorna cell line; Dr , P. Chany, Institut de Recherehes Scientifiques sur le Cancer, Villejuif, France, for supplying the LF Wistar hepatoma cell line; Professor P. Padieu, University of Dijon, for supplying the 18 PC-FR Wistar fetalliver cellline; Dr. J. Desgres, Faculte Mixte de Merleeine et de Pharmacie, Dijon, for controlling the metabolis m of progesterone into 6-alpha-hydroxypregnenolone by cell lines of liver origin; Dr. C. Samarut, Höpital Edouard Herriot, Lyon, for doing the erythrocyte-antibody rosette assay; and Professor H. Hirai and Dr. S. Nishi, both of the Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan, for providing the test system for rat (lfetoprotein. We thank Mrs. L. Saint- Vincent and Miss C. Drevon, both of the IARC, for technical help; Dr. T. Kuroki, Dr. J. P. Lamelin, and Dr. G. Lenoir, all of the IARC, and Professor J. P. Revillard, Höpital Edouard Herriot, for useful discussions; Dr. C. A. LinselI, IARC, for his constant support; and Miss S. Reynaud, IARC, for typing this manuscript. 1
2
MATERIALS AND METHODS Animals.-BD IV and BD VI rats were used. They have been maintained inbred since 1970 by systematic brother X sister matings at the International Agency for Research on Cancer in Lyon. Cell cultures.- The rat cell lines and tissue material used are listed in tables 1 and 2. They included epitheVOL. 60, NO. 1, JANUARY 1978
lial-like cell lines IAR 2 and IAR 20 derived from livers of 10-day-old BD VI rats and IAR 6 derived from the liver of an 8-week-old BD IV rat, as weIl as corresponding malignant cell lines obtained either by spontaneous malignant transformation or after in vitro treatment with DMN or MNNG (9-11). IAR 6-1 RT-7 was derived from a tumor induced in a BD IV rat by ip injection of the IAR 6-1 cell line. We verified that the malignant liver cell lines originated in the liver by noting the appearance of a rat liver-specific membrane antigen (12) and by controlling the capacity of the cell lines to metabolize the progesterone in 6-alpha-hydroxy-pregnenolone (13, 14). The malignancy of transformed cell lines was verified by their sc or ip injection into syngeneic newborn rats (10). Cell lines were maintained in tissue culture flasks (Falcon Plastics, Oxnard, Calif.) with Williams-D medium supplemented with 10% heatinactivated fetal bovine serum (Flow Laboratories, Rockville, Md.). They were serially passaged after 0.25% trypsin dissociation.
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Xenogeneic antisera.-Rabbits were immunized with cultured living cells suspended by trypsinization. They received three to five ip injections of 1-3 x 107 cells in 2 ml phosphate-buffered saline every 2 weeks and were bled 10 days after the last injection. The antibodies directed against the normal antigenic components of rats were eliminated by in vivo absorption (15). Syngeneic male rats were given ip injections of 0.1-10 ml rabbit antiserum per kilogram body weight and were bled by cardiac puncture 16 hours later. Negative controls were given injections of normal rabbit serum. To determine whether the positive immunofluorescence reactions corresponded to individual or cross-reacting antigens, additional absorptions were done in vitro as follows: Living cells were suspended at a final concentration of 5x 107/ml of serum previously absorbed in vivo. After incubation for 2 hours at 37° C, the cells were removed by centrifugation. The in vitro absorptions were each performed three times . Sera from pregnant rats.-Blood was taken at day 1520 of gestation or 7 days after the last delivery from pregnant BD rats having had at least 3 litters. Negative control sera were obtained from virgin female rats. Membrane immunojluorescence tests.-Immune sera were assayed against target cells by indirect membrane immunofluorescence, as described by Martin et al. (8). Fluorescein isothiocyanate conjugates had the following origin: Anti-rabbit IgG sheep serum came from the Institut Pasteur (Paris, France), and anti-rat IgG goat serum was purchased from H yland Laboratories (Los Angeles, Calif.). Membrane immunofluorescence was
examined with a Tiyoda fluorescence microscope. Fluorescence indexes were calculated as the percentage of cells unstained by negative control serum minus the percentage of cells unstained by the test sera divided by the percentage of cells unstained by negative control serum (16). The percentages of cells unstained by negative control serum and by fluorescence reagent alone were over 99.5%. RESULTS Reaction 01 Xenogeneic Antisera Absorbed In Vivo
The reactions of xenogeneic antisera after in vivo absorption are indicated in table 1. Of 7 rabbit antisera, 3 reacted with the corresponding immunizing celllines (IAR 6-1, IAR 2-31, and IAR 20 PCI-3). The reactivity of anti-IAR 6-1 was restricted to the isologous cell line and to the derived IAR 6-1 RT-7 cell line. However, anti-IAR 2-31, in addition to reacting with the immunizing cell line, reacted with several malignant cell lines derived from IAR 2 and 6, transformed either chemically or spontaneously. Similarly, anti-IAR 20 PCI-3 reacted not only with the isologous cell line but also with malignant cell Iines derived from IAR 2; it did not react with the corresponding nonmalignant cell lines. None of the antisera reacted with normal rat cells derived from various organs, with cells taken from 10-, 15-, and 19-day fetuses (table 2), with intestinal carcinoma cells of BD IX rats, or with liver tumor cell Iines from Wistar rats (table 1).
TADLE l.-Membrane immunofluorescence ofvarious rat cells incubated with in vivo absorbed sera ofrabbits immunized against rat liver cells chemically transformed in vitro Fluorescence indexes" with antisera to: Original rat strain
BDIV BDIV BDVI BDVI BDIV BDIV BDIV BDVI BDVI BDVI BDVI BDIX Wistar Wistar BDIV BDVI Wistar a b C
d
e f
J
Target cells a Non transformed IAR6 IAR 6-10 IAR2 IAR20 Chemically transformed IAR 6-1 IAR 6-1 RT-7 d IAR 6-4 IAR 2-22 IAR 2-28 IAR 2-31 IAR 20 PCl-3 e DHD intestinal carcinoma/ LF hepatoma f D 202 hepatoma/ Spontaneously transformed IAR 6-7 IAR 2-25 18 PC-FR
Treatment of cell by"
IAR 6-1
IAR 6-1 IAR 6-4 RT-7
IAR 222
IAR 228
IAR 231
IAR20 PCl-3
Nothing MNNG Nothing Nothing
0.02 0.00 0.00 0.02
0.00 0.00 NT NT
NT 0.00 NT NT
0.00 NT 0.00 0.00
NT NT 0.00 0.00
0.07 0.00 0.03 0.00
0,00 0.00 0.12 0.09
DMN DMN DMN DMN MNNG MNNG MNNG DMH DAB DAB
0.97 0.92 0.00 0.00 0.02 0.00 0.01 0.00 0.00 0.00
0.00 0.05 0.00 NT NT 0.00 0.00 NT NT NT
0.00 0.00 0.00 NT NT 0.00 0.00 NT NT NT
0.00 NT 0.00 0.02 0.00 0.00 0.00 NT NT NT
0.00 0.00 NT NT 0.02 0.00 0.00 NT NT NT
0.55 0.82 0.85 0.85 0.82 0.93 0.12 0.00 0.00 0.00
0.00 0.00 0.19 0.88 0.82 0.79 0.88 0.00 0.00 0.00
Nothing Nothing Nothing
0.06 0.00 0.00
0.00 NT NT
0.00 NT NT
0.00 0.00 NT
0.00 0.00 NT
0.55 0.86 0.05
0.00 0.82 0.02
All celllines were ofliver origin except the intestinal carcinoma cellline that was derived from a tumor induced by DMH. DMH = 1,2-dimethylhydrazine; DAB =p-dimethylaminoazobenzene. A fluorescence index ofO.30 was considered positive. NT=not tested. Celllines derived from a tumor induced in a BD IV rat given an ip injection oflAR 6-1. Celllines derived from MNNG treatment oflAR 20PC, a cloning cellline from IAR 20. Celllines established from tumors.
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TUMaR-SPECIFIC ANTIGENS ON RAT LIVER CELLS TRANSFaRMED IN VITRa
TABLE 2.-Membrane immunofluorescence staining o{ cells {rom [reshly dissociated rat tissues incubated with in vivo absorbed sera o{ rabbits immunized against rat lioer cells chemically trans{ormed in »itro Fluorescence indexes with antisera to:" Rat strain
Age
Origin of target cells IAR 6-1
BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BD VI BD IV and BDVI BDVI BDVI BDVI
10-day fetus 15-day fetus
Whole-body tissue Liver Whole-body tissue Liver Whole-body tissue Liver Spleen Intestine Lung Kidney
19-day fetus 12-wk-old rat
IAR 6-1 IAR 6-4 RT-7
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 NT 0.00 NT NT NT NT NT NT
IAR222
IAR228
IAR231
IAR 20 PCI-3
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 NT 0.00 NT NT NT NT NT NT
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
" N'I'r-not tested.
Reactions of Xenogeneic Antisera Absorbed In Vivo and In Vitro
Ta determine how many antigenic systems were present, the three antisera were absorbed further with isologaus and/or homologous cell lines (table 3). The TABLE 3.-Demonstration of tumor-specific individual and crossreacting antigens on malignant rat liuer cells by membrane immunofluorescence with rabbit antisera absorbed in vivo and in uitro Fluorescence indexes with:" Antisera to
In vitro absorption with"
IAR 6-4
IAR IAR 2-22 2-31
0.97 0.00 0.00 0.92
0.00 NT NT NT
0.00 NT NT NT
0.00 NT NT NT
0.01 NT NT NT
Nothing IAR 6-1 (DMN) IAR 2-22 (DMN) IAR 2-31 (MNNG) IAR 20 PCI-3 (MNNG) Fetal bovine serum
0.55 0.00 0.00 0.00 0.26 0.55
0.82 0.00 0.00 0.00 0.24 0.76
0.85 0.00 0.00 0.00 0.29 0.85
0.93 0.00 0.00 0.00 0.33 0.88
0.12 NT NT NT 0.00 NT
Nothing IAR 6-1 (DMN) IAR 2-31 (MNNG) IAR 20 PCI-3 (MNNG) Fetal bovine serum
0.00 0.00 0.00 0.00 NT
{Nothing IAR 6-1
IAR 2-31
IAR 20 PCI-3
IAR 20 PCI-3
IAR 6-1
IAR6-1 (DMN) IAR 6-1 RT-7" IAR 2-31 (MNNG)
0.19 0.00 0.00 0.00 NT
0.88 0.83 0.00 0.00 NT
0.79 0.75 0.00 0.00 NT
0.88 0.80 0.00 0.00 0.88
In vitro absorption was done three times with 5x lO' cells or 0.2 ml fetal bovine serum/ml of antiserum, b NT=not tested. c Cell Iine derived from a tumor induced in a BD IV rat given an injection of IAR 6-1.
31 could be eliminated with malignant ceillines divided from IAR 2 and 6; thus tumor-specific cross-reacting antigen(s) was expressed on these cell lines. Since the reactivity of anti-IAR 20 pe 1-3 was completely abolished by malignant cell lines derived from IAR 2 and 20, another tumor-specific cross-reacting antigen(s) was expressed on these ceillines.
Reactions of Sera From Multiparous Pregnant Rats Of the 7 assayed sera from multiparaus pregnant rats, 3 (#368, 555, and 395) reacted with freshly dissociated fetal cells from whole-body and liver tissues of 15-day-old BD IV arid/or BD VI rats with fluorescent indexes greater than 0.30 (table 4). However, they did not react with the 3 malignant IAR cell lines that carried either tumor-specific individual or cross-reacting antigens as evidenced by their reaction with heterologous sera.
DISCUSSION On the basis of other studies, we discarded the possibility that membrane immunofluorescence reacTABLE 4.-Membrane immunofluorescence o{cells [rom. uihole-body and lioer tissues o{ 15-day rat [etuses and [rom in oitro chemically transformed rat li ver cell lines after incubation with sera front multiparous pregnant rats Fluorescence indexes with:" Serum No.
No.of pregnancies
Rat strain
a
reactivity of anti-IAR 6-1 could be eliminated only with isologous cell lines IAR 6-1 and IAR 6-1 RT-7; therefore , only tumor-specific individual antigen(s) was expressed on these cell lines. The reactivity of anti-IAR 2-
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4 3 4 4
BD VI
5
BDIV
IAR cells chemically transformed in vitro
Fetal cells"
4 4
BD IV
BD VI
6-1
RT-7 2-31
NT NT 0.26 0.16 NT 0.30 0.12
0.13 0.00 0.33 0.40 0.08 0.48 0.17
NT NT 0.00 0.00 NT 0.00 0.00
NT NT 0.00 0.00 NT 0.00 0.00
NT NT 0.00 0.00 NT 0.00 0.00
20 PCI-3 NT NT 0.00 0.00 NT 0.00 0.00
" NT=not tested. b Freshly dissoeiated cells.
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YOKOTA, SIZARET, AND MARTEL
tions observed in this study were due to the binding through Fe receptors of nonspecific immune complexes present in absorbed rabbit antisera for the following reasons: a) Heat-aggregated immunoglobulins were not bound by the malignant cells (17), and b) malignant cells did not form erythrocyte-antibody rosettes when incubated with ox red blood cells coated with rabbit immunoglobulins (18). We concluded that the rat liver celilines transformed in vitro by chemical carcinogens acquired two different types of tumor-specific antigens: One type was absent from other malignant cell lines and was therefore considered to be an individual turnor-specific antigen; the other type was found on several malignant cell lines and was therefore considered to be tumor-specific crossreacting antigen. Oncofetal antigens have been detected by xenogeneic antisera on rat sarcoma cells (1) and on rat intestinal cells (8) from chemically induced tumors. Moreover, with the use of rat immune material such as lymphoid cells or sera from multiparous rats (6, 7), oncofetal antigens have been detected on a wide range of cells from chemically induced tumor. Techniques of assays included toxicity or membrane immunofluorescence. The scarcity of reports of oncofetal antigens on cell lines transformed in vitro (19) can be contrasted to the frequency of re ports of such antigens on cell lines derived from tumors. The present results are in line with the contrasting findings of those reports: Since the antigens we studied were not detectable on fetal cells taken at several stages of development and since none of the malignant cell lines reacted with sera from multiparous pregnant rats, these antigens seem to be different from oncofetal antigens. As expected, the nature of tumor-specific cross-reacting antigen(s) was not related to the ehernieals used for transformation; however, it seemed somehow related to the original cell line, as if only certain cell lines possess specific repressed potentialities that are realized only after malignant transformation. Moreover, because these antigens were not detected on malignant liver cell lines derived from Wistar rats transformed either in vitro or in vivo, they showed specificity to BD IV and BD VI rats. In this respect, these antigens differ from the PNantigen (chemically induced in vivo) detected by Okita et al. (20) on malignant hepatocytes and from the common antigen detected by Steele and Sjögren (21) on colon carcinoma cells. They appear to be of the same nature as the antigens, which were cross-reactive and which differed from the embryonie antigen, detected by Iype et al. (5) on rat liver cells transformed in vitro with chemical carcinogens. Since the techniques required for the study of tumorspecific transplantation antigens and tumor-specific surface antigens differ, we do not know whether individual tumor-specific surface antigens are identical to tumorspecific transplantation antigens. The observation in our study that only 1 of 6 malignant cell lines exhibited tumor-specific individual antigen can be explained by the use of xenogeneic antisera. Compared to homoloJ
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gous antisera, such antisera may be less likely to pick up small antigenic differences such as "individual malignant antigens." Another possible explanation is that , since most of our experiments were done on cell lines of polyclonal origin, individual antigenic determinants varied from cell to cell. The multiplicity of such determinants could result in antigenic competition. This may explain why tumor-specific cross-reacting antigens are more easily detected on malignant cell lines of polyclonal origin. Another possible reason that the antigens were not detected is the dilution of antisera during the in vivo absorption. It seems unlikely that the antigens detected in this study are induced by a sarcoma virus since, with the use of an antiserum against the gs-30 antigen of murine oncornavirus, no reaction was observed on any of the chemically transformed malignant cell lines and since none of our antisera reacted with the 3T3-FL celiline expressing virus antigens of murine oncornavirus after infection by the Gross virus. In addition, the supernatants of malignant cell lines did not induce the malignant transformation of any of the four original cell lines. As could be expected from the lack of reaction of rabbit anti-turnor-specific sera with rat fetal liver cells, the three antigens were found to be different from rat a-fetoprotein, since none of the malignant cell lines reacted by immunofluorescence with rabbit anti-rat afetoprotein. Further experiments will be necessary to ascertain whether the antigens reported have a role in the immune defense of the host.
REFERENCES (1) THoMsoN DM, ALEXANDER P: A cross-reacting embryonie anti-
(2)
(3)
(4) (5)
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(10)
gen in the membrane of rat sarcoma cells which is immunogenic in the syngeneic host. Br] Cancer 27:35-47, 1973 ZOLLER M, PRICE MR, BALDWIN RW: Cell-mediated cytotoxicity to chemically-induced rat tumours. Int] Cancer 16:593-606, 1975 MONDAL S, IYPE PT, GRIESBACH L, et al: Antigens of cells derived from mouse prostate cell after malignant transforrnation in vitro by carcinogenic hydrocarbons. Cancer Res 30:1593-1600,1970 EMBLETON M], HEIDELBERGER C: Antigenicity of clones of mouse prostate cells transformed in vitro. Int] Cancer 9:8-18, 1972 IYPE PT, BALDWIN RW, GLAVES D: Cell surface antigenic changes induced in normal adult rat liver cells by carcinogen treatment in vitra. Br J Cancer 27:128-133,1973 BALDWIN RW, GLAVES D, VaSE BM: Ernbryonie antigen expression in chemically induced rat hepatomas and sarcomas. Int J Cancer 10:233-243, 1972 STEELE G], SJÖGREN HO: Embryonie antigens associated with chemically induced colon carcinomas in rat. Int J Cancer 14:435-442, 1974 MARTIN F, KNOBEL S, MARTIN M, et al: A carcinofetal antigen iocated on the membrane of cells from rat intestinal carcinoma in culture. Cancer Res 35:333-336, 1975 MONTESANO R, SAINT-VINCENT L, TOMATIS L: Malignant transformation in vitro of rat liver cells by dimethylnitrosamine and N-methyl-N'-nitro-N-nitrosoguanidine. Br J Cancer 28:215-220, 1973 MONTESANO R, SAINT-VINCENT L, DREVON C, et al: Production of epithelial and mesenchymal tumours with rat liver cells transformed in vitro. Int] Cancer 16:550-558,1976 VOL. 60, NO. 1, JANUARY 1978
TUMOR-SPECIFIC ANTIGENS ON RAT LIVER CELLS TRANSFORMED IN VITRO (11) KUROKI T, DREVON C, SAINT- VINCENT L, et al: Studies on the use of liver parenchymal cell in in vitro carcinogenesis. In Comptes-Rendus of the Centre National de Recherche Seientifique, Menton meeting, 1976. In press (12) !KAWA Y, NIWA A, TOMATIS L, et al: Transformation of a rat liver celliine by murine sarcoma virus. Proc Am Assoc Cancer Res 14:109, 1973 (13) DESGRES J, GUIGUET M, BEGUE RJ, et al: Etude du metabolisme he pato-foetal de la progesterone a l'aide de cellules hepatiques de rat en culture. In Comptes Rendus: Organisation des Laboratoires - Biologie Prospective: 3e Colloque de Pont-aMousson, 1975. Paris: Expansion Scientifique Francaise , 1975, pp 663-670 (14) - - - : Study of progesterone metabolism in foetal and postnatalliver cells in culture. In Advances in Mass Spectrometry, Biochemistry and Medicine (Frigerio A, Castagnoli N, eds). New York: Spectrum, 1976 (15) HERSEY P: The protective effect of antisera against leukaemia in
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vivo. A reappraisal. Br J Cancer 28:11-18,1973 (16) KLEIN E, KLEIN G: Antigenic properties of lymphomas induced by the Moloney agent. J Natl Cancer Inst 32:547-568, 1964 (17) DICKLER HB: Studies of the human lymphocyte receptor for he at aggregated or antigen-complexed immunoglobulin.J Exp Med 140:508-522, 1974 (18) BROCHIER J, SAMARUT C, REVILLARD JP: Rosette technique for identification of human mononuclear cells bearing Fe receptors. Biomedicine 23:206-209, 1975 (19) EMBLETON MJ, HEIDELBERGER C: Neoantigens on chemically transformed cloned C3H mouse embryo cells. Cancer Res 35:2049-2055, 1975 (20) OKITA K, KUGMAN LH, FARBER E: A new common marker for premalignant and malignant hepatocytes induced in the rat by chemical carcinogens. J Natl Cancer Inst 54: 199-201, 1975 (21) STEELE G JR, SJÖGREN HO: Cross-reacting tumor-associated antigen(s) among chemically induced rat colon carcinomas. Cancer Res 34:1801-1807,1974
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