Characterization of a Human Chorionic Gonadotropin-Producing Testicular Choriocarcinoma Cell Line

0022-534 7/87 /1373-0548$02.00/0 THE JOURNAL OF UROLOGY

Vol. 137, March

Copyright © 1987 by The Williams & Wilkins Co.

Printed in U.S.A.

CHARACTERIZATION OF A HUMAN CHORIONIC GONADOTROPINPRODUCING TESTICULAR CHORIOCARCINOMA CELL LINE HARUKI YAMAZAKI,* SHIGEYUKI KOTERA, HIROSHI ISHIKAWA, AND TOYOHEI MACHIDA From the Department of Urology and Department of Anatomy, The Jikei University School of Medicine, Tokyo, Japan

ABSTRACT

A testicular choriocarcinoma cell line designated JHTK-1 was established in vitro from a mixed germ cell tumor xenografted into nude mice, and may prove to be the first human choriocarcinoma cell line of testicular origin in long-term culture. The cells have been kept in culture for three years and produce human chorionic gonadotropin persistently. The chromosome number distribution ranged from tetraploidy to septaploidy, and trypsin G-band karyotyping showed that all the chromosomes were of human origin. Dibutyryl cyclic adenosine monophosphate at a concentration of 1 mM caused more than 15-fold stimulation of human chorionic gonadotropin secretion with morphological alteration in JHTK-1 cells. This suggests that dibutyryl cyclic adenosine monophosphate induces the differentiation of cytotrophoblastic cells into syncytiotrophoblastic cells among JHTK-1 cells. When JHTK-1 cells were heterotransplanted into the subcutis of BALB/c nude mice, large cystic tumors were produced with histological characteristics similar to choriocarcinoma. Testicular choriocarcinoma is a highly aggressive and lethal germ cell tumor of the testis. The establishment of testicular choriocarcinoma cell lines is desired in order to provide experimental models for the investigation of the basic biology of this tumor. Although a number of testicular germ cell tumor cell lines have been previously established, these seem to consist mainly of embryonal carcinoma cells and do not persistently produce tumor markers such as a-fetoprotein (AFP) or human chorionic gonadotropin (HCG) in long-term culture. 1 - 3 In this study, we cultivated HCG-producing cells cloned from a mixed germ cell tumor xenografted into nude mice and established a HCG-producing testicular choriocarcinoma cell line, which was designated JHTK-1. We describe the method of culture and cytobiological characteristics of this cell line in the present report. MATERIALS AND METHODS

Culture materials and cell culture. JHTK-1 was derived from a testicular mixed germ cell tumor taken from a 45-year-old male. The serum levels of AFP, HCG, and HCG-,B subunit were all elevated preoperatively, being 460 ng./ml., 210 mIU/ml., and 5.3 ng./ml., respectiv.ely. Orchiectomy v:as performed on May 6, 1981. Histologically, the tumor was mainly embryonal carcinoma mixed with syncytiotrophoblasts and intratubular malignant germ cells. Prior to in vitro culture, xenograft tumors were developed by implanting the excised tumor into BALB/c nude mice (Clea Japan, Inc., Tokyo). The xenograft tissue was minced with a razor blade in a medium containing 1,000 PU dispase/ml. (Godo-Syusei Co., Tokyo). The dissociated cells were resuspended in a growth medium containing anti-nude mouse rabbit serum (1:20) to eliminate the nude mouse mesenchymal cells immunologically4 and placed in 6-cm. Falcon plastic dishes (Falcon Plastics, Oxnard, Calif.) in five per cent CO 2 at 37C. The growth medium used was Ham's F-10 medium containing 20% fetal calf serum (Flow Lab., McLean, Va.), 50 U penicillin and 50 µg. streptomycin/ml. The cells grew confluently after five to seven days. Then, cloning of the cells was performed using a modified Rose chamber as described previously. 5 Briefly, single cells were removed with a micropipet and each Accepted for publication September 30, 1986. * Requests for reprints: Department of Urology, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan.

plated into 0.55% soft agar in the upper compartment of a modified Rose chamber. Cells from the same culture were placed in the lower compartment. The growth medium was perfused through the upper compartment and then the lower compartment. The single cell in the upper compartment gradually proliferated and developed into a colony of cells, which constituted a clone. Several clonal strains were thus established. Of these, the JHTK-1 strain produced a high level of HCG. JHTK-1 was grown in 25-cm. 2 Falcon flasks with seven ml. of culture medium and incubated in five per cent CO 2 at 37C. The cultures were fed two to three times weekly and split every five to six days using 0.25 per cent trypsin and 0.02 per cent ethylenediaminetetracetic acid (EDTA) in Ca++ free-phosphate buffer saline. Growth rate. The growth curve of the JHTK-1 cell line was determined at the 55th passage using 30-mm. plastic dishes. Chromosome analyses. Chromosome analyses were performed at Special Reference Laboratory, Tokyo. The cells at passage 103 were treated with 0.1 µg./ml. of colcemid for four hr. at 37C, placed in a hypotonic solution (0.075 M KCl) for 15 min., and fixed with methanol-acetic acid (3:1). They were then heated and treated with 0.025% trypsin, stained with Giemsa, and analyzed. The chroinosone number distribution was determined by examining 100 cells in metaphase. Seven cells were analyzed for trypsin G-band karyotyping. Electron microscopy. Cells were fixed for 16 hr. in 2.5 per cent glutaraldehyde solution made with 0.1 M phosphate buffer, pH 7.4, at 4C, and washed with five per cent sucrose in phosphate buffer. Cells were postfixed in two per cent osmium tetroxide in phosphate buffer at 4C for two hr., dehydrated with ethanol, embedded in a mixture of epon-araldite, stained with lead citrate and uranyl acetate, and then observed by electron microscopy (JOEL JEM lOOS). Marker proteins. The concentrations of HCG, HCG-,B, pregnancy- specific ,81 -glycoprotein (SPl), human placental lactogen (HPL), AFP, and carcinoembryonic antigen (CEA) in the culture media and fetal calf serum were measured by radioimmunoassay (RIA) at Special Reference Laboratory, Tokyo. The RIA for HCG (heterologous HCG RIA, CIS) showed crossreactivity (at B/B = 50%) with HCG-,B (280%), and that for HCG-,B (homologous HCG-,B RIA, CIS) exhibited minimal cross-reactivity with complete HCG (2%). Effect of dibutyryl cyclic adenosine monophosphate on JHTK1 cells. JHTK-1 cells at the 50th and 105th passages, plated at

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a of about 5 treated with LO mM (Sigma v1.,nun,cu After 12, 24, hr. of treatment, the conditioned media were collected for HCG measurement, and the cells ,vere counted. Heterotransplantation. About 2 X 106 cells at the 40th passage were injected subcutaneously into six-week-old BALB/c nude mice. The tumors that developed were removed and fixed with 10 per cent formalin for histological examination.

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RESULTS

JHTK-1 cells grew well in culture and have been successfully maintained through 120 passages for three years. Morphological characteristics of cells. JHTK-1 cells are epithelial cells with prominent cytoplasmic granules, which grow in single and multiple layers as tightly packed colonies (fig. 1). Under electron microscopy the cytoplasm contained a few mitochondria, poorly developed rough endoplasmic reticulum, and many free polysomes. Microvilli were observed on the cell surfaces (fig. 2). These electron microscopic observations corresponded to the characteristics of cytotrophoblasts. Growth characteristics. The growth curve of JHTK-1 cells is shown in fig. 3. The population doubling time was calculated to be about 24 hr. Chromosome analyses. The chromosome numbers ranged from 107 to 141 without distinct modal number (fig. 4). Trypsin G-banding study showed that there were only human chromosomes and that there were extensive structural abnormalities. Structural changes identified as lp-, , 13p+,

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Days after Plating FIG. 3. Growth curve of JHTK-1 cells at 55th passage. Cells were removed every day from triplicate dishes, and average cell counts were determined.

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FIG. 2. Electron micrograph of JHTK-1 cells (Xl0,000).

romosorne FIG. 4. Distribution of chromosome number of JHTK-1 cells at 103th passage

were seen to be common to all the cells chromosomes were also observed; some of these were likewise common to all the cells studied (fig. HCG and were detected persistently in the culture media of the JHTK-1 cells during serial passage. The levels of HCG and HCG-{3 in the media reached approximately 800 mIU /ml. and 30 ng./ml., respectively, at the 10th and 75th passages, and approximately 250 mIU /ml. and 5.0 ng./ml., respectively, at the 90th and 110th passages. On the other hand, SPl, HPL, AFP and CEA were not detected in the media. It was confirmed by similar RIAs that the fetal calf serum used (Flow Lab. lot 29101959) did not contain detectable quantities of HCG or HCG-(3. Effect of DBcAMP on JHTK-1 cells. Upon addition of one mM DBcAMP to the JHTK-1 cultures at the 50th and 105th passages, significant morphological and biochemical changes occurred. Morphologically the cells developed many short processes, occasionally becoming asteroid in shape within 48 hr.

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(fig. 6). Biochemically the addition of 1 mM DBcAMP increased HCG secretion more than 15-fold in the culture media. DBcAMP also inhibited cell proliferation. The changes produced by DBcAMP at the 105th passage are shown in fig. 7. It should be noted that these changes in morphology and HCG secretion occurred almost simultaneously. Heterotransplantation. Eight out of 12 nude mice implanted with 2 x 106 cells developed subcutaneous tumors of cystic form. By seven weeks, the mean tumor size was 2.8 x 2.5 X 1.3 cm., the mean tumor weight was 867 mg., and the mean volume of the cyst fluid was 7. 7 ml. The levels of the marker proteins (mean± SD) in the cyst fluid were as follows: HCG, 190,000 ± 7,075 mlU/ml; HCG-/J, 3,500 ± 1,244 ng./ml.; SPl, 24.0 ± 7.4 ng./ml.; HPL, 0.13 ± 0.05 µg./ml. AFP and CEA were not detected. Histologically, the tumor was choriocarcinoma in which cytotrophoblastic cells and syncytiotrophoblastic cells were intermingled (fig. 8).

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FIG. 6. Effect of DBcAMP (1 mM) on morphology of JHTK-1 cells within 48 hr. of treatment. Inverted photomicrograph (xlOO).

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FIG. 7. Effect of DBcAMP on production of HCG [A] and cell proliferation [BJ of JHTK-1 cells at the 105th passage. Conditioned media were collected each time from triplicate dishes for HCG assay, and cells were counted. DBcAMP, 1 mM dibutyryl cyclic adenosine monophosphate; CONT, control with no additions.

DISCUSSION

We established a choriocarcinoma cell line designated JHTK-1, which was isolated from a mixed germ cell tumor of the testis xenografted into nude mice. JHTK-1 is of completely human origin and a clonal cell line, since trypsin G-band karyotypic analysis showed that this cell line was composed strictly of cells with human chromosomes and showed no evidence of murine chromosomes, and since various common structural abnormalities were seen in all the cells studied (fig. 5).

The most characteristic function of choriocarcinoma, which is a malignant derivative of placental trophoblast, is the production of placental proteins such as HCG, SPl, and HPL. In

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FIG. 8. Tumor in nude mice showing histological features of chorio-

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cells grown under routine conditions also this hA~~.Ar, some syncytial cells are believed to present among the JHTK-1 cells. Addition of DBcAMP to the cultures inhibited cell proliferation, increased the number of differentiated cells and increased HCG secretion by more than 15-fold at the same time (fig. 7). Using morphology and HCG secretion as criteria for identification of differentiation, we found that the cells that appeared upon treatment with DBcAMP seemed to be syncytial cells. Thus, these results suggested that DBcAMP chemically induced or accelerated the cytologic differentiation of actively proliferating cytotrophoblastic cells into HCG-producing syncytiotrophoblastic cells. The cell line JHTK-1 should provide an excellent in vitro model system for testicular choriocarcinoma. It will be valuable for basic studies on testicular choriocarcinoma concerning hormone secretion, differentiation, sensitivity to anticancer agents, and generation of monoclonal antibodies. This line may lead to the development of new and improved therapeutic modalities for testicular choriocarcinoma. REFERENCES

routine cultures, JHTK-1 cells secreted a significant level of HCG but undetectable levels of SPl and HPL. Upon heterotransplantation, the level of HCG became highly elevated, with slight increases in the SPl and HPL levels in the cyst fluid of the tumor. In contrast, AFP was detected neither in vitro nor in vivo, These results indicated that JHTK-1 is choriocarcinoma uncontaminated yolk sac tumor cells. It is known that DBcAMP stimulates HCG secretion in cultures of normal trophoblast and gestational choriocarcinoma cells and may have an effect on their morphology and cell growth as well. 6 JHTK-1 cells also showed sensitivity to DBcAMP. In routine cultures, the cell population of JHTK-1 appeared almost homogeneous, and cells of different morphology were rarely seen. Upon addition of DBcAMP, pronounced morphological changes occurred (fig. 6). The altered cells closely resembled the "broad and elongated cells" showing cytoplasmic projections and interconnection of adjoining cells, which Pattillo et al. 7 described in their trophoblast cultures and proved to be associated with HCG production. Syncytial cells synthesize and secrete HCG. Since JHTK-1

Williams, R. D., Bronson, D. L., Elliott, A. Y., Lane, P. H. and Fraley, E. E.: Human testicular cells in culture. Urol. Clin. North Am., 4: 529, 1977. 2. Williams, R. D.: Human urologic cancer cell lines. Invest. Urol., 1.

17: 359-363, 1980. 3. Wang, N., Trend, B., Bronson, D. L. and Fraley, E. E.: Nonrandom abnormalities in chromosome 1 in human testicular cancers. Cancer Res., 40: 769, 1980. 4. Okabe, T., Suzuki, A., Ohsawa, N., Kosaka, K. and Terasima, T.:

Immune elimination of host fibroblasts for the cultivation of human tumors transplanted into nude mice. Cancer Res., 39: 4189, 1979.

5. Ishikawa, H., Shiino, M., Arimura, A. and Edward, G. R.: Functional clones of pituitary cells derived from Rathke's pouch epithelium of fetal rats. Endocrinology, 100: 1227, 1977. 6. Hussa, R. 0., Pattillo, R. A., Ruckert, A. C. F. and Scheuermann, K. W.: Effects of butyrate and dibutyryl cyclic AMP on hCGsecreting trophobiastic and non-trophoblastic cells. J. Clin. Endocrinol. Metab., 46: 69, 1978. 7. Pattillo, R. A., Smith, T. C., Delfs, E. and Mattingly, R. F.: Cytologic and hormonal activity of trophobiast in explant culture. Am. J. Obstet. Gynecol., 96: 337, 1966.