Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8211-8215, November 1988 Immunology are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid line (/malignant lymphoma/patient sera) YUKIMASA SHIRAISHI* AND HIROAKI SOMAt *Laboratory of Cell Biology, Department of Anatomy, Kochi Medical School, Nankoku-City 781-51, Kochi, Japan; and tDepartment of Obstetrics and Gynecology, Tokyo Medical College, Tokyo, Japan Communicated by David Harker, August 1, 1988 (receivedfor review March 12, 1987)

ABSTRACT We have cloned malignant cells carrying [malignant lymphoma (ML), ovarian cancer (OVC), stomach specific antigens associated with ovarian cancer (OVC) and cancer (ST), or hepatic cancer (HC)]. We have developed a malignant lymphoma (ML) from BS-SHI.4M cells, a line simple method ("panning") for the separation of malignant derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B- clones with the corresponding cancer activity from lymphoblastoid cell line isolated from a patient with Bloom the heterogeneous population of cells originating from car- syndrome. Since BS-SHI-4M cells react with sera from various cinogen-treated BS-SHI-4 cells. It is based on the observa- individual cancer patients at relatively low frequencies (2-9%), tion by Catt and Tregear (4) and Wysocki and Sato (5) that as detected by an indirect immunofluorescence technique, cell antibody molecules adsorb onto polystyrene surfaces and clones that specifically react with sera from patients with OVC bind antigen. We coated polystyrene dishes with the diluted and ML were separated by the "panning" method in which sera from OVC or ML patients and permitted cells with polystyrene dishes were coated with sera from OVC and ML cancer antigens to bind to the coated dishes. The panning patients and cells with the corresponding antigens bound to the process was repeated twice and we have cloned independent dishes. Subsequent cloning by limiting dilution provided cell malignant clones carrying cancer antigens that consistently clones highly enriched for OVC- and ML-associated antigens. reacted with sera from OVC or ML patients and that, Karyotype analyses revealed that cell clones with OVC and ML therefore, may be useful for clinical diagnosis of cancer. antigens had common marker chromosomes, der(14)t(14;14) Further cell cloning by limiting dilution provided pure cell (pll;qll),t(6;?)(p25;?) and t(9;?)(q34;?), besides t(17;?) clones with OVC- and ML-associated antigens from the (q25;?) found in the OVC-antigen-positive clones and t(5;?) carcinogen-treated BS B-LCL. We have also analyzed kary- (pl3;?),t(7;?)(q36;?) found in the ML-antigen-positive clones. otypes in cell clones with OVC- and ML-associated antigens Interestingly, in cell clones with a strong OVC antigen re- and discussed the to the sponse, the distal part ofthe Y chromosome (Yqll) was missing marker chromosomes relating in 100% ofthe cells. Therefore the cell line BS-SE114M appears expression of these cancer antigens. to be a reservoir of cell clones each of which carries a specific MATERIALS AND METHODS tumor antigen and thus provides a potential tool for rapid Cell Line and Cell Culture. The permanent BS B-LCL, serological diagnosis of cancer. termed BS-SHI-4, was established from a BS patient as described (6, 7). BS-SHI-4 cells were found to have increased Bloom syndrome (BS) is an autosomal-recessive genetic SCE (sister-chromatid exchange) levels (70.4 + 2.64 ex- disorder characterized by stunted growth, sensitivity to changes) in cells labeled with bromodeoxyuridine for two cell sunlight, immunodeficiency, and marked predisposition to cycles. Although chromosome instability, including breaks various types of cancer (1). BS differs distinctly from certain (5-9o) and quadriradials (0.01-0.03%), was detected at a other recessive genetic disorders that are also characterized relatively low frequency, the karyotype 46,XY was basically by chromosome instability and cancer proneness in that it is normal. Tests of cell surface markers, such as clumping of not predisposed to a particular type of cancer, as seen in lymphoid cells, no spontaneous rosette formation, and the xeroderma pigmentosum (a predisposition to cancer of sun- presence of surface immunoglobulin (IgM), revealed, the exposed tissues), Fanconi anemia (a predisposition to leuke- lymphocytes to be of B-cell origin (8, 9). The cells were mia and hepatic cancer), and ataxia telangiectasia (a predis- maintained in RPMI 1640 medium supplemented with 10% position to lymphoma) (1). The striking feature of BS is that (vol/vol) fetal calfserum at 37°C in a humidified 5% C02/95% it predisposes patients to diverse types of . This air incubator. suggests that in BS the types of cancer are basically of the Malignant Transformation. Either 107 BS-SHI-4 cells or same kind as in normal persons but that the process of 107 normal (KS86) cells were exposed to 1-methyl-3-nitro-1- is accelerated. Therefore, the elucidation of nitrosoguanidine (MeNNG) at 0.3 ,ug/ml for 24 hr, washed the mechanism(s) of carcinogenesis in BS would be of great with fresh RPMI 1640 medium, and then reincubated in RPMI value in the study of human carcinogenesis in general. 1640 medium supplemented with 10% (vol/vol) fetal calf B-lymphoblastoid cell lines (B-LCLs) have been established serum at 37°C in a 5% C02/95% air incubator for 2-3 weeks. from BS patients that are highly susceptible to malignant Detection of Antigens Associated with Cancer. MeNNG- transformation after carcinogen treatments, as shown by treated BS-SHI-4 cells were suspended by pipetting and were tumor production in nude mice and cell growth in agar (2, 3). washed twice in phosphate-buffered saline (PBS; 0.01 M When BS-SHI-4 cells, a BS B-LCL, were treated with a sodium phosphate, pH 7.2/0.15 M NaCl). Cell surface or carcinogen and cultured in vitro, the resulting cells (BS-SHI- cytoplasmic antigens were examined with fresh cell suspen- 4M) carried cancer antigens that reacted with the antibodies sions or fixed cell-smear preparations, respectively, by present in the sera of patients with various malignancies Abbreviations: BS, Bloom syndrome; B-LCL, B-lymphoblastoid The publication costs of this article were defrayed in part by page charge cell line; MeNNG, 1-methyl-3-nitro-1-nitrosoguanidine; OVC, ovar- payment. This article must therefore be hereby marked "advertisement" ian cancer; ML, malignant lymphoma; ST, stomach cancer; HC, in accordance with 18 U.S.C. §1734 solely to indicate this fact. hepatic cancer; SCE, sister chromatid exchange. 8211 Downloaded by guest on September 26, 2021 8212 Immunology: Shiraishi and Soma Proc. Natl. Acad. Sci. USA 85 (1988) Table 1. Characterization of B-LCLs isolated from normal (KS82) and BS (BS-SHI-4) blood samples after carcinogen treatment Surface Cytoplasmic Carcinogen, immunoglobulin, % immunoglobulin, % Cell line 0.3 Ag/ml y a A y a A SCE, no. Karyotype KS82 None 30 0 0 32 0 0 4.6 ± 0.04 46,XY KS82 MeNNG 28 0 0 31 0 0 4.7 ± 0.05 46,XY BS-SHI-4 None 0 0 56 0 0 59 70.8 ± 2.86 46,XY BS-SHI-4M MeNNG 0 0 51 0 0 48 71.6 ± 2.46 46,XY/abnormal clones For surface and cytoplasmic immunoglobulin, values shown are average values of >500 cells for y, a, and /A chains. SCE values are expressed as mean ± SEM. indirect immunofluorescence with fluorescein isothiocya- normal individual (KS82) and a BS patient (BS-SHI-4), before nate-conjugated goat antisera monospecific for human IgG and after carcinogen treatment, is shown in Table 1. When the (10-12). For the detection of cell surface antigens, 3 x 106 carcinogen-treated BS-SHI-4 and KS82 cells were cultured in cells, washed with PBS twice, were suspended in 500 ,ul of vitro for 14-21 days and fresh and fixed cells were treated with sera from cancer patients diluted 1:30 in PBS and incubated serum from various cancer patients (15 OVC patients, 12 ML at 4°C for 90 min. Cells were then washed with cold PBS patients, 15 ST patients, and 15 HC patients) by the indirect twice, incubated with fluorescein isothiocyanate-conjugated immunofluorescence technique, positive immunofluorescence goat antisera monospecific for human IgG (1:40 dilution with was detected only in MeNNG-treated BS-SHI-4 cells, al- PBS) at 4°C for 90 min, washed with cold PBS twice, and though the positive cell frequencies were low and varied from sealed under nonimmunofluorescent glycerin and a cover- 2% to 9.9% (Fig. 1). BS-SHI-4, KS82, and MeNNG-treated slip. Immunofluorescence was viewed by using an Olympus KS82 showed no positive reaction to serum from any cancer fluorescence microscope with epi-illumination. patient. The finding that MeNNG-treated BS-SHI-4 cells Separation ofCells with Cancer Antigens by Using a Panning (BS-SHI-4M) did not react with the sera from normal subjects Procedure. Polystyrene bacteriological Petri dishes (100 x 15 but reacted only with sera from cancer patients strongly mm) were used in all experiments. Serum from a patient was indicates that these cells possess antigens associated with diluted 1:30 in PBS, and 10 ml was poured immediately onto cancer. The frequency of the cancer-antigen-positive cells in the plates. The solution was swirled on each plate until the cultures of BS-SHI-4M cells has remained at a constant level bottom surface was evenly covered. After 20 hr at 4°C, the for >2 years, indicating that this cell line may provide a stable serum was decanted, and the dishes were washed three times supply of cells with cancer antigens. with Hanks' balanced salt solution (HBSS) and treated with Panning with Serum from OVC and ML Patients. It was 5 ml of HBSS supplemented with 5% (vol/vol) fetal calf uncertain (i) whether the cancer antigens carried by the serum for 1 hr at room temperature. About 2 x 107 nucleated BS-SHI-4M cells (as revealed by the use of serum from cells were suspended in 5 ml of HBSS plus 5% (vol/vol) fetal various cancer patients) were common to all the cancers calf serum and poured onto each of the dishes coated with tested or (ii) whether they were specific for each kind of serum (antibody), with care taken to avoid bubbles. The cancer and, therefore, serum from a given cancer patient plates were incubated on a level surface at room temperature would react with only a small fraction ofthe cells tested. Low for 60 min, then the unattached cells were redistributed by and variable frequencies of immunofluorescence-positive tilting and swirling the plate, and the unadhered cells were cells with specific serum from various cancer patients (Fig. 1) removed with a pipette. Plates were washed by gently would be consistent with the latter hypothesis. Thus poly- pouring 5-10 ml of HBSS down the side of the dish and then styrene bacteriological Petri dishes (100 x 15 mm) were swirling, tilting, and decanting. To recover the bound cells, coated with serum from OVC and ML patients that bound the plate was filled with 10 ml of RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum, and the BS-SHI-4 entire surface of the plate was flushed by using a Pasteur pipette. Cells were then transferred to 6-cm culture dishes and, cultured at 37°C in a humidified 5% C02/95% air incubator. The panning process was 10 performed again by cn using serum from another patient. U) Cell Cloning by Limiting Dilution. BS-SHI-4M cells, which had been selected twice by the panning procedure with serum 0.a) from two patients, were cloned by the limiting dilution BS-SHI-4M method. Cells were seeded in a microculture plate (96 wells), 0. 0 in which 10, 30, 50, and 100 cells were placed in each well. _o Constant cell growth was observed in microculture plates 10 containing >50 cells per well. These cell clones were allowed to proliferate and then used for cancer antigen examination and for karyotype analysis. Chromosome spreads were 0. A prepared by a routine air-drying method (13). Differential N ML OVC ST HC staining of sister chromatids and karyotype analysis were Antiserum performed by using the fluorescence plus Giemsa (FPG) technique (14) and G- and Q-banding, respectively. For SCE FIG. 1. BS-SHI-4 and BS-SHI-4M cells (fixed cells) were incu- analysis, the cells were labeled with bromodeoxyuridine at 10 bated with sera diluted 1:100 from 10 normal subjects (N), 12 ML, 15 ,ug/ml for 48 hr. OVC, 15 ST, and 15 HC patients, and antibody binding to the cytoplasm ofcells was detected by indirect immunofluorescence. No positive reaction was detected between BS-SHI-4 cells and any ofthe RESULTS sera used, 2-9o of the BS-SHI-4M cells were positive when Cancer-Associated Antigens in Carcinogen-Treated BS-SHI-4 incubated with sera from patients with ML, OVC, ST, and HC, and Cells. Characterization of the B-LCLs established from a BS-SHI-4M cells did not react with normal sera. Downloaded by guest on September 26, 2021 Immunology: Shiraishi and Soma Proc. Natl. Acad. Sci. USA 85 (1988) 8213 BS-SHI-4M cells, and BS-SHI-4M cells were incubated in response with ML sera. As seen from Fig. 2, there is no large these dishes. The cells that adhered to the dish contained 10- difference in the immunofluorescence-positive percentage 30% positive cells by immunofluorescence after one panning among the sera from 15 OVC patients [including those with procedure. After a second panning procedure (with serum serous and mucinous (papillary) adenocarcinomas] and sera from another patient), the percentage ofpositive cells rose to from 12 ML patients (6 with B-cell type and 6 with T-cell type 40-50%o and 30-50% with OVC and ML sera, respectively. In ML). Fig. 3 a and b shows cytoplasmic immunofluorescence the first and second panning procedures, the cell population from BS-SHI-4M cells and clones derived from this line after that did not adhere to the coated plate contained almost no panning and subsequent limiting dilution cloning. Fig. 3c cells that were antigen positive to the sera type used in the shows no immunofluorescence for BS-SHI-4M-OV1OV2C1 panning. Fig. 2 shows the percent ofBS-SHI4M-derived cell cells incubated with normal serum (1:100 dilution with PBS). clones that were immunofluorescent-positive with the sera Detection of membrane-associated antigens was performed from 15 OVC, 12 ML, 15 ST, and 15 HC patients. These by an indirect immunofluorescence method. BS-SHI-4M- clones were designated as BS-SHI4M-OV1 and BS-SHI-4M- OV1OV2C1 and BS-SHI-4M-ML1ML2C1 cell lines expressed ML1 (clones from one panning procedure with OVC1 and membrane antigens that reacted with antibodies in the sera of ML1 sera, respectively) and as BS-SHI-4M-OV1OV2 and OVC and ML patients, respectively (Fig. 4). A large fraction BS-SHI-4M-ML1ML2 (clones from a second panning proce- of viable cells in these cell lines was positive for antigens, dure with OVC2 and ML2 sera, respectively) (Fig. 2). Cells although the intensity of fluorescence varied among different that were most proficiently cloned and that had antigens with sera-e.g., OVC1, OVC2, OVC5, OVC9, OVC11, ML', ML2, the most specific reactivity were selected for further cloning ML8, and ML12 sera gave brilliant fluorescence. The relative by limiting dilution in 96-well plates. Cell clones obtained intensities ofthis membrane antigen staining were comparable from this procedure were designated as BS-SHI-4M- to that of cytoplasmic fluorescence. None of the normal sera OV10V2C1 and BS-SHI-4M-MLjML2C1 and gave a 70-90% and nonmalignant patient sera reacted with the BS-SHI-4M- positive response with OVC sera and a 60-70% positive OV10V2C1 and BS-SHI-4M-MLML2C1 cell lines. These two

60 BS-SHI-4M-OV1 BS-SHI-4M-ML,

40

20

BS-SHI-4M-OV1OV2 BS-SHI-4M-ML, ML2 60

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80 it'

60 40~~~~~~~& A

A A

j40 A

f 9

20 N ML OVC ST HC N ML OVC ST HC Antiserum FIG. 2. BS-SHI-4M-OV1, BS-SHI-4M-ML1, BS-SHI-4M-OV1OV2, BS-SHI-4M-ML1ML2, BS-SHI-4M-OV1OV2C1, and BS-SHI-4M- MLjML2C1 cells were incubated with sera as in Fig. 1. A, Cytoplasmic immunofluorescence; A, surface antigen. Downloaded by guest on September 26, 2021 8214 Immunology: Shiraishi and Soma Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 3. (a) Antigen-antibody reaction between BS-SHI-4M-OV1 cells and OVC1 serum. (b) Antigen-antibody reaction between BS-SHI-4M-OVIOV2CI cells and OVC3 serum. (c) Negative reaction between BS-SHI-4M-OV1OV2C1 cells and a normal serum. All antibody binding to cytoplasm was detected by indirect immunofluorescence. cell clones are clearly specific to the kind of sera used in the - 17, - 18, - 20, + 22,del(2)(q?),t(5;?)(pl3;?),t(6;?)(p25;?), panning process to separate the clone from the general t(7;?)(q36;?),t(9;?)(q34;?), + der(14)t(14;14)(pll ;qll), + marl, population ofMeNNG-treated BS-SHI-4M cells: BS-SHI4M- + mar2, + mar3. Karyotype analysis of BS-SHI-4M- OV1OV2C1 cells did not react with serum from ML, ST, and OV1OV2C1 cells (97 cells) showed that 82 cells were 47, HC patients, whereas BS-SHI-4M-ML1ML2Cj cells did not X,del (Y) (qil), + 12, - 13,- 14, - 16,del(2) (q?),t(6;?) (p25;?), react with serum from OVC, ST, and HC patients (Fig. 2). t(9;?)(q34;?), + der(14)t(14;14)(pll;qll),t(17;?)(q25;?), Chromosome Analysis in Cloned Cells with OVC and ML + mar3, + mar4 (Fig. 5 Lower), 10 cells were 45,X,del(Y)(qll), Cancer Antigens. Chromosome analysis performed with G- - 14, - 18, - 20,t(6;?)(p25 ;?),t(9;?)(q34;?), + der(14)t(14;14) and Q-banding techniques showed a basically normal kary- (pll;qll),t(17;?)(q25;?), + mar3, and 5 cells were 44,X,del otype (46,XY) in the original BS-SHI-4 cell line, although (Y)(qll), -14, - 18, - 20,t(6;?)(p25;?),t(9;?)(q34;?), chromosome instabilities including breaks (5-8%) and quadri- +der(14)t(14;14)(pll;qll). The widespread presence of these radials (0.01-0.02%) were detected at relatively low fre- markers strongly suggests the single-cell origins of BS-SHI- quency. 4M-OV1OV2C1 cells and BS-SHI4M-MLjML2Cj cells. Cell BS-SHI-4M cells, resulting from MeNNG-treatment of clones that did not adhere to the dish when BS-SHI-4M cells BS-SHI-4 cells, showed karyotype abnormalities involving were panned with OVC sera contained a normal Y chromosome certain marker chromosomes [t(6;?)(p25;?),t(9;?)(q34;?), structure (with Y-body) in addition to these marker chromo- der(14)t(14;14)(pll;qll),t(17;?)(q25;?)], even though the un- gomes. Thus, BS-SHI4M cells that retained the distal Y chro- treated cell line had basically a normal karyotype (46,XY). mosome (qll-12) had no detectable OVC antigen activity. Karyotype analysis of 50 BS-SHI4M-MLML2C1 cells (Fig. The common marker chromosomes in BS-SHI-4M- 5 Upper) revealed the following karyotype: 46,XY, - 6, - 14, OVIOV2C1 and BS-SHI-4M-ML1ML2C1 cell lines were

FIG. 4. (a and b) Antigen-antibody reaction between BS-SHI-4M-OV1OV2CI cells and OVC1 serum. (c and d) Antigen-antibody reaction between BS-SHI-4M-MLIML2Cl cells and a ML serum. Cell surface antibody binding was detected by indirect immunofluorescence. Downloaded by guest on September 26, 2021 Immunology: Shiraishi and Soma Proc. Natl. Acad. Sci. USA 85 (1988) 8215 the OVC antigen clone and to t(5;?)(pl3;?) and t(7;?)(q36;?) 0 O.* 4?b '. xi in the ML antigen clone. More interestingly, the BS-SHI- 0 it 4!ii I 11 4M-0V1OV2C1 cell clone with strong antigen expression was missing the distal part of the Y chromosome del(Y)(qll-qter) 1 4 2 3 5 in 100% of the >1000 cells examined. BS-SHI-4M cells that did not adhere to the OVC sera-coated polystyrene dish had i A 4 I I A a normal Y chromosome with Y body and had no detectable (i i OVC antigen activity. The finding that the proximal part of 6 7 8 9 4- the Y chromosome (male-determining part) suppresses the i OVC antigen is interesting, considering that the BS-cell- 6 donor patient was a male. Our previous results (3) indicate 13 14 Ml 2M3 6 1 7 18 that BS cell lines with high SCE levels are highly susceptible MI; to malignant transformation by chemical carcinogenesis, when SCEs exceeded 140 SCEs per cell (over the baseline of -2 -70 SCEs per cell). The appearance ofkaryotypic changes in 19 20 21I Y BS-SHI-4M cells and their -derived clones (BS-SHI-4M- 0V10V2Cj and BS-SHI-4M-ML1ML2C1) may reflect phe- nomena secondary to malignant transformation or may have 20 ~ played a key role in the expression of the antigens associated r )5 with the cancers. 1 2 3 4 OVC is the most lethal ofall gynecological cancers (15, 16). Most ovarian tumors are epithelial, predominately serous and ovarian 7 9.0 mucinous cystadenocarcinomas. The detection of }l tumors is often fatally delayed because of their location and 1 lack of diagnostic methods for early detection; early diagno- sis would greatly improve the prognosis for patients, which a 7.I I8 is the reason why attempts have been made to detect tumor 13 14 I16 17 18 markers for diagnosis and therapeutic monitoring. ML is also known as a widely distributed lymphoid malignancy charac-

* a terized by histological, immunological, and cytogenetical heterogeneity. Although ordinary testing procedures with 19 20 m3m4 21 22 x Y monoclonal antibodies distinguish between B and T lympho- cytes, the identification ofthis malignancy has been difficult. FIG. 5. Karyotypes of clones BS-SHI-4M-MLjML2C1 (Upper) Our protocol would enable detection of the malignancy of and BS-SHI-4M-0V10V2C1 (Lower). both T-cell and B-cell MLs, and Hodgkin ML with only the use of serum from the patient. Diagnostic procedures with t(6;?)(p25;?),t(9;?)(q34;?),der(14)t(14;14)(pll;qll) and mar3. serum from the patient would be the most convenient for Cell clones with no cancer antigen activity did not have the early diagnosis. der(14)t(14;14)(pll;qll) marker, although many subclones were obtained from the effluent populations. The authors are grateful to Miss Michiyo Ozawa for her technical assistance. This work was supported by grants-in-aid from the DISCUSSION Nissan Science Foundation and from the Ministry of Education, We have detected stable expression of antigens associated Science and Culture of Japan. with cancer on the cell surface of a carcinogen-transformed BS B-LCL, BS-SHI-4, by using diluted serum from various 1. German, J. (1974) in Chromosomes and Cancer, ed. German, cancer and immunofluorescence. In con- J. (Wiley, New York), pp. 601-607. patients indirect 2. Shiraishi, Y. (1985) EMBO J. 4, 2553-2560. trast, most of the other B-LCLs established from BS patients 3. Shiraishi, Y., Yosida, T. H. & Sandberg, A. A. (1985) Proc. during the last 3 years retained a high level of SCE and were Nail. Acad. Sci. USA 82, 5102-5106. highly susceptible to malignant transformation when their 4. Catt, K. & Tregear, G. W. (1967) Science 158, 1570-1571. SCE rate increased above a certain critical level (3, 6, 7). The 5. Wysocki, L. J. & Sato, V. L. (1978) Proc. Nail. Acad. Sci. expression of cancer antigens from carcinogen-treated BS USA 75, 2844-2848. type-III cells with surface IgG seemed unstable and limited to 6. Shiraishi, Y., Kubonishi, I. & Sandberg, A. A. (1983) Cancer the antigen associated with lymphoid malignancies. Thus Genet. Cytogenet. 56, 129-138. BS-SHI-4M cells, derived from MeNNG-treated BS-SHI-4 7. Shiraishi, Y., Yoshimoto, S., Miyoshi, I., Kondo, N., Orii, T. and express cancer & Sandberg, A. A. (1983b) Cancer Res. 43, 3836-3840. cells, apparently readily stably various 8. Platts-Mills, T. A. E. & Ishizaka, K. (1975) J. Immunol. 114, antigens that were studied by indirect immunofluorescence 1058-1063. with antibodies present in sera of cancer patients. Even 9. Jondal, J. & Klein, G. (1975) J. Exp. Med. 138, 1365-1378. though the initial frequency of antigen-positive cells was low 10. Minowada, J., Janossy, G., Greaves, M. F., Tsubota, T., at 2-9% in the MeNNG-treated BS-SHI-4M cells, this fre- Sahai, Srivastava, B. I., Morikawa, S. & Tatsumi, E. (1978) J. quency rose to 70-90% for the OVC antigen (BS-SHI-4M- Nail. Cancer Inst. 60, 1269-1277. 0V10V2C1 cells) and 60-80% for the ML antigen (BS-SHI- 11. Greaves, M. F., Brown, G. & Rapson, N. T. (1975) Clin. 4M-ML1ML2Cl cells) after two panning cycles and a subse- Immunol. Immunopathol. 4, 67-84. quent cloning with limiting dilution. These enrichment 12. Greaves, M. F. (1975) Prog. Hematol. 9, 255-303. the of the cell anti- 13. Shiraishi, Y., Freeman, A. I. & Sandberg, A. A. (1976) Cyto- procedures clearly increased specificity genet. Cell Genet. 17, 162-173. gen-serum antibody reaction (Figs. 2 and 3), suggesting it 14. Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158. might be usable for OVC and ML diagnosis. Karyotype 15. Heinonen, P. K., Tontti, K., Koivula, T. & Pystynen, P. (1985) analyses revealed that cell clones with OVC and ML antigens Br. J. Obstet. Gynaecol. 92, 528-531. had common markerchromosomes, t(6;?)(p25;?),t(9;?)(q34;?) 16. Mattes, M. J., Cordon-Cardo, C., Lewis, J. L., Old, L. J. & and der(14)t(14;14)(pll;qll), in addition to t(17;?)(q25;?) in Lloyd, K. 0. (1984) Proc. Natl. Acad. Sci. USA 81, 568-572. Downloaded by guest on September 26, 2021