[CANCER RESEARCH 35, 2025-2032, August 1975]

SUMMARY

A gastric choriocarcinoma cell line synthesizing humanchorionic gonadotropin (HCG) was established in 1971 byOboshi et a!. and was found to possess human placentalalkaline phosphatase. The present paper also deals with therelationship between the cell growth and HCG secretion andwith cellular localization of HCG and human placentalalkaline phosphatase by cytochemical and ultrastructuralmethods. This cell line was found to secrete HCG duringcellular proliferation, with the maximum secretion in thestationary phase (about I zlU/cell/48 hr), and the hormonecould be detected in a small proportion of mono- and/ormultinuclear cells in both logarithmic and stationaryphases. The organ-specific, heat-stable, L-phenylalaninesensitive, immunoreactive human placental alkaline phosphatase was localized on the cell membrane of many cells.Ultrastructurally, the line consisted mainly of cytotrophoblastic and intermediate cells in the process of syncytialformation, with more or less squamous metaplasia. Fromthese findings it was concluded that the cell line maintainedthe properties of trophoblastic cells from morphological andfunctional aspects, i.e., it was a cell line with two distinctmarker substances.

INTRODUCTION

A hormone-synthesizing human trophoblastic cell linehas been established and maintained in vitro for more than 5years by Pattillo et a!. (24). To the best of our knowledge, 4other well-authenticated cell lines ( 13, 2 1, 25, 29) derivedfrom genital and extragenital trophoblastic tumors have sofar been reported.

The purpose of this report is to characterize a cell line,SCH [which was established by Oboshi et a!. in 1971 andreported in 1972 (21)], in relation to its growth pattern,morphology, and production of HCG2. This line wasderived from an extragenital choriocarcinoma, and it main

1 This work was supported in part by Grants 90398 and 801071 from

the Japanese Ministry of Education.2 The abbreviations used are: HCG, human chorionic gonadotropin;

HPALP, human placental alkaline phosphatase; PAS, periodic acidSchiff; PFA, paraformaldehyde; FITC, fluorescein isothiocyanate.

Received January 1, 1975; accepted April 2, 1975.

tamed trophoblastic characteristics in regard to morphologyand hormone production. Functionally, this cell line produces 2 easily detectable markers, HCG and HPALP. Thiscorrelates well with morphological differentiation from thecytotrophoblast (or trophoblastic stem cell) to the syncytiotrophoblast. The fact that the source of the cell line was thestomach (probably gastric mucosa) indicates “dysdifferentiation― of the gastric epithelial tumor cells to the trophoblastic cells. The reappearance in vitro of epithelial cellssimulating gastric mucosal cells has not yet been confirmedby this investigation.

MATERIALS AND METHODS

Cell Line. Cell line SCH, established by Oboshi et a!. (21)from a metastatic omental lesion of gastric choriocarcinoma from a 46-year-old male, was used. The histology ofthe original tumor revealed features characteristic of achoriocarcinoma coexisting with a small area of adenocarcinoma producing mucin. High titers ofurinary HCG (from8,000 to 32,000 IU) were noted until death. The autopsyrevealed, in addition, gynecomastia and Leydig cell hyperplasia.

This cell line had been serially transferred withoutdispersing agents before the 59th passage. The specimensfor this study were obtained from the cells of the 62nd to76th passages.

Culture Medium. Roswell Park Memorial Institute Medium 1640 (Nissui Chemicals, Tokyo) with 20% fetal calfserum was the culture medium used. The medium wassupplemented by penicillin, 100 units/mi, and streptomycm, sg/ml.

Cell Cultivation for Growth Kinetics. After being dispersed with a mixed solution of 0. 1% trypsin and 0.02%EDTA, the cell suspension of the 62nd passage containing 2x l0@cells was inoculated into plastic Petri dishes (60 mmin diameter; Falcon Plastics, Oxnard, Calif.) and grown byreplicate culture (5) in a humidified atmosphere containing5% CO2 and 95% air. Twenty-four hr after the transfer, and

every 48 hr thereafter, 2 or 3 dishes were harvested forcalculation of the cell number. Culture media were storedfor HCG assays. The media in the rest of the dishes werereplaced with fresh ones. By using a Burker-TUrk counterplate, the number of cells of each dish was calculated 4times. The means were determined for each dish and then

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A Human Gastric Choriocarcinoma Cell Line with Human ChorionicGonadotropin and Placental Alkaline Phosphatase Production'

Toru Kameya, Hiroyuki Kuramoto, Kenji Suzuki, Takiko Kenjo, Teruyuki Oshikiri, HiroatsuHayashi, and Motoyoshi Itakura

Pathology Division, National CancerCenterResearchInstitute, Tsukiji 5-chome,Chuo-ku, Tokyo 104[T. K.]; Department of ObstetricsandGynecology, Kitasato University School of Medicine, Kanagawa [H. K.]; and Department of Obstetrics and Gynecology, Keio University School ofMedicine, Tokyo [K. S., T. K., T. 0., H. H., M. I.], Japan

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T. Kameya el a!.

for every harvest. They then were plotted on a semilogarithmic graph.

Assay of HCG. The HCG titers in the culture medium ofeach dish were assayed in duplicate with radioimmunoassay(I, 17) using the double-antibody method (18). The detection range was between 1.25 and 640 mIU/mI. For eachharvest, the mean ±S.D. was calculated and plotted. Thefresh medium, in which no HCG was detected, was used as acontrol.

Cell Cultivation for Cytology and Cytochemistry. Approximately 1 x l0@cells were inoculated into a square tube (40x 17 x 8 mm in size; MA-8 Miharu, Chiba) which containeda 32- x 12-mm coverslip. Following 4 to 20 days of culture,the cells in sheets or piled-up aggregates on a coverslip werefixed for morphological examinations.

Methods for Cytological and Cytochemical Examinations.For Giemsa staining, the specimens were fixed with ethanolfor 10 to 20 mm. Usual stainings were also performed forneutral fat (Sudan III), glycogen (PAS) before and afteramylase digestion, and mucins (Alcian blue-PAS or mucicarmine) after paraformaldehyde fixation. For light microscopic enzyme histochemistry, the cells on the coverslipwere fixed with 4.0% PFA or Baker's formol calcium (3) for5 to 30 mm, and after washing in gum-sucrose solution, the

enzyme reaction for alkaline phosphatase was made by amodified Burstone's azo dye method, as previously described in detail (10). The procedures for heat stability andL-phenylalanine inhibition tests have also been described(10). Also, search was made for minimal concentrations ofL-phenylalanine needed for complete inhibition by testingconcentrations from 0.001 to 0.05 M.

For electron microscopic localization of alkaline phosphatase, after fixation with 2.5% glutaraldehyde followed bywashings with Tris-maleate buffer, a cell sheet was scrapedoff the coverslip, cut into pieces, and incubated in asubstrate solution at room temperature. The incubationmedium, which was prepared by the method of Mayahara eta!. (14), contained lead citrate. After incubation, thespecimens were fixed in 1% 0504 buffered with cacodylateand processed for usual Epon embedding.

For immunohistochemistry, the coverslips were washedwith a serum-free culture medium. A brief fixation (10 to 30mm) with 4% PFA was performed. For intracellularlocalization of HCG and HPALP, the indirect methodsusing peroxidase-labeled (19) or FITC-labeled (8) sheepanti-rabbit -y-globulin antibodies were used. The quality ofrabbit anti-HPALP was previously reported (10). Thespecificity of the rabbit anti-HCG was confirmed byOuchterlony's diffusion plate method. The anti-rabbit “yglobulin (the 2nd antibody) (10) was conjugated with horseradish peroxidase (type VI; Sigma Chemical Co., St.Louis, Mo.), by Avrameas' glutaraidehyde (2) or byKawaoi's peroxidase aldehyde (1 1) method. The conjugateswere kindly supplied by Dr. T. Mukojima of the NationalCancer Center Hospital.

For light microscopy using the peroxidase-labeled antibody method, the cells on the coverslip were counterstainedfor nuclei with 0. 1% methyl green, after immunostaining.

For electron microscopy, after immunostaining, the spec

imens were processed, as described in the enzyme histochemistry method, when the cell sheet was not tightlyadhered to the glass surface. When cells were tightlyadhered to the slip, osmification and dehydration wereperformed on cells attached to the glass surface. The Eponembedding was conducted by inverting a plastic capsulefilled with Epon mixture on the coverslip, followed bycuring at 60°.After hardening of the Epon mixture, the cellsheet, together with the capsule, was taken off the coverslipin a cooled cryostat room (below —20°).

Ultrathin sections of the cell sheet or pellet were madewith an LKB or Porter-Blum ultramicrotome equipped witha diamond knife. Electron microscopic observations wereconducted first without and then with metal counterstainings.

For the FITC-labeled antibody method, the cell sheet onthe coverslip was fixed with ethanol, acetone, or PFA for 10to 30 mm. Immunostainings were done as usual. The slipswere mounted in a 10% glycerol solution and observed byOlympus, Chiyoda, or Leitz fluorescence microscope, andphotographs were taken with high-speed Ektachrome (Kodak) films.

The following controls were used for immunostainings:(a) normal rabbit ‘y-globulinor serum, followed by the 2ndconjugated antibody, (b) rabbit anti-porcine glucagon oranti-bovine ACTH antibodies (unrelated antibodies), folIowed by the 2nd conjugated antibody, and (c) anti-H PALPor anti-HCG, followed by the nonlabeled 2nd antibody, andthen by the 2nd conjugated antibody (inhibition test). In thecase of peroxmdase immunostaining, the diaminobenzidinereaction for peroxidase without antigen-antibody reactionwas essential as a control because there were frequentlynonspecific background stainings of diaminobenzidine.

RESULTS

Growth Kinetics. The growth curve of each subculture forthe experiment could be divided into lag, logarithmicgrowth, and stationary phases, respectively. On the 1st day,the number ofliving cells was calculated as 17.5 x 104/dish,indicating 87.5% of plating efficiency. The logarithmic

5 10 15 :ioDAYS AFTER SEEDING

Chart I. The growth curve of SCH cells. It can be divided into lag,logarithmic growth, and stationary phases. The population-doubling timeat the logarithmic growth phase was calculated to be 67.5 hr.

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Choriocarcinoma Cell Line

growth, noted on approximately the 5th day of culture, wascomparatively slow and steady. As shown in Chart 1, itspopulation-doubling time was counted to be 67.5 hr. As theculture continued, the cell sheets were easily detached fromthe bottom of the culture dish.

HCG Secretion and Growth Kinetics. At every harvest,the amount of HCG in culture media was plotted per l0@cells (Chart 2). On the 1st day of subculture, the titer washigh, showing almost the maximum value per cell (Chart 2).Thereafter, the amount showed sudden decline, reachingalmost zero in the early logarithmic phases. Subsequently,in the middle of the logarithmic phase, a small amount ofHCG was detected and, as the culture continued, renewedsynthesis and discharge of the hormone at a surprisinglyhigh rate were again noted; the doubling time of HCGsecretion was much shorter than that of growth. Thesecretion/dish maintained a high level and continued,although its rate became slow. On the other hand, theamount of secretion/cell became almost constant after thecell growth reached the stationary phase. At this point, thevalue was calculated to be I jzIU/48 hr, which was themaximum throughout the whole cycle (Chart 2).

Routine Light Microscopy. SCH cells appeared epithelialand polygonal, with a pavement-like monolayer arrangement and a piling-up growth in later phases. The cellcontained large, sometimes giant, nuclei with increased andunevenly distributed chromatin and one or a few prominentnucleoli. Mitotic figures were frequent (Fig. I). In theabundant cytoplasm, vacuoles were frequently noted, occasionally displacing nuclei. Multinuclear giant cells were notrare.

Ultrastructure. Cells were connected to one another withnumerous typical desmosomes (Fig. 2). Both free andadjoining surfaces were often equipped with prominentmicrovilli (Figs. 3, 4, and 12) with complex interdigitations,forming widened intercellular spaces (Fig. 3).

Many nuclei were indented, with distinct nuclear pores,coarsely granular peripheral chromatin concentration, and

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0

C,

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I0C

DAYS AFTER SEEDING

Chart 2. The growth curve and HCG secretion per I0@SCH cells. Theamount of HCG was assayed every 48 hr. The secretion sharply declined atthe lag phase and became undetectable on the 7th day or at thegrowth-initiating point. Then, at the logarithmic-growth phase, a steeplyincreasing amount of HCG was noted: it had a doubling rate that was muchhigher than that ofthe cell growth, with the maximum rate (I @zIU/cell/48hr) at the late stationary phase.

prominent nucleoli. Multinucleated cells were occasionallyfound. Many cells contained abundant free ribosomes, oftenwith a form of polysomes, and poorly developed rough andsmooth endoplasmic reticulum with some smooth-surfacedvesicles and vacuoles (Fig. 2). In this type of cells, welldeveloped Golgi complexes were rare (Fig. 4). Many cellswere full of tonofilaments or bundles of thick tonofibrils(Fig. 3) often converging to desmosomes, simulating cells ofstratified squamous epithelium. A few cells, however,possessed moderately or well-developed dilated or shortlystacked endoplasmic reticulum (Fig. 5) and small Golgielements resembling intermediate (27) or syncytiotrophoblastic cells. No mucinous vacuoles, zymogen-, or endocrine-type secretory granules were seen. A considerablenumber of lipid vacuoles were frequently found in thecytoplasm (Figs. 2 and 4). A particular feature worthy ofnote was the presence of occasional “isolated―or intracytoplasmic desmosomes that were not connected with the cellsurface membrane at either end (Fig. 4). In any specimen,especially that of later stages, a large cytoplasmic defect wasoften encountered. This may correspond to the light microscopic finding of cytoplasmic vacuoles displacing nuclei tothe periphery. A small percentage of the cells containedaggregated glycogen particles (Figs. 6 and 7).

Cytochemistry. By light microscopy, PAS-positive materials that were digested with amylase were present in mostof the cells (Fig. 6). Frequently, they also contained fatdroplets positively stained with Sudan III. No staining wasseen by Alcian blue or mucicarmine.

With the azo dye method for alkaline phosphatase, therewas a considerable cell population that showed a stronglypositive reaction on the cell membrane (Figs. 8 and 9). Theperipheral region of the piled-up cells disclosed a particularly intense reaction (Fig. 9), but the central part of the cellaggregate showed a rather weak reaction (Fig. 9). Thereaction was not significantly decreased after heating of thespecimen at 65°for 15 or 30 mm (Fig. 9). The alkalinephosphatase activity was completely inhibited with 0.05 ML-phenylalanine (Fig. 10) and largely inhibited with 0.01 M,in contrast to the corresponding concentrations of Dphenylalanine. Ultrastructurally, the reaction was exclusively localized on the free surface, including that ofmicrovilli (Figs. 3, 4, and 7). However, not all free surfaceswere positive (Fig. 4), and most complex interdigitations ofthe cellular membrane, widened intercellular gaps, andother organelles lacked enzyme reaction (Fig. 3). By usingthe peroxidase- (Fig. 11 and 12) or FITC-labeled antibodymethod, the reaction products to anti-HPALP were localized on the same sites as those of enzyme histochemistry, byboth light and electron microscopy. All controls revealednegative reactions in corresponding locations. However,cells undergoing degeneration (e.g., vacuolated cells detached from the coverslip) showed a nonspecific diffusebrown staining of diaminobenzidine in the cytoplasm withno preferential staining of the cell membrane, which alsotook place with normal rabbit 7-globulin instead of antiHPALP.

As for HCG localization, only a small proportion ofmono- and probably multinuclear cells showed intense

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granular positive reaction products in the cytoplasm withthe FITC-labeled method (Fig. 13), although positivelystained cells increased in number in the stationary phase. Itwas noteworthy that the peripheries of the areas of piled-upcells were more frequently occupied by positively stainedcells as compared with the wide zone of monolayer sheets.The immunostaining was successful only when the specimens were vigorously washed with the buffered solution andthe 1st antibody was diluted with a serum-free culturemedium. Immunostaining for HCG by the peroxidaselabeled method was not satisfactory in cultured cells, whilesyncytial cells in tissue sections of normal chorionic villi,hydatidiform moles, and choriocarcinoma were very wellstained.

DISCUSSION

Oboshi el a!. (21) established the in vitro cell line SCHfrom gastric choriocarcinoma and have maintained the cellsystem for more than 3 years. They reported that this cellline possessed characteristic features of cytotrophoblasticcells with a slight degree of differentiation to syncytial cellsand was capable of producing immunologically and biologically active HCG.

In this study, a large amount of HCG in the culturemedium just after the inoculation was considered to havebeen released from cells that had been maximally synthesizing and storing HCG at the stationary phase. This assumption was confirmed when the amount of HCG decreasedrapidly after the transfer to an undetectable level. The cellsdid not have the ability to synthesize HCG during the periodbetween the transfer and the initial part of the logarithmicgrowth phase. Thereafter, cells began to secrete HCG withincreasing synthesizing activity. This suggests that cellulardifferentiation begins not after the cessation of growth, butalong with active growth. The maximum secretion wasnoted in the period when the growing ability was reduced.Cells under these circumstances can obviously differentiate,because they do not spend much energy on their growth(13). Consequently, it can be concluded that the HCGsecretion of the cell line is not independent of its growth.Concerning HCG secretion, the level in the SCH line wasapproximately 20% of that of Pattillo's BeWo line (24) and5 times as much as Kohler's JEG-4 line (13), which was the

least of his 6 clonal lines.Abundant free ribosomes, a moderate amount of glyco

gen, and poorly developed organelles are common featuresof cytotrophoblasts. On the other hand, moderately developed, dilated endoplasmic reticulum, microvilli, isolateddesmosomes, and occasional lipid vacuoles are consideredto be features characteristic of intermediate cells (22, 27)between the cyto- and syncytiotrophoblasts. The isolateddesmosomes, described previously as intrasyncytial cleftswith desmosomes, pairs of electron-dense rods, etc., byother authors (4, 7, 22, 27) are regarded as a terminalprocess of syncytium formation. However, typical syncytialcells were rarely seen in this cell line, in which cytotrophoblastic and intermediate cells were main components.Intracytoplasmic defects observed represented some types

of degenerative process in the cell. These morphologicalfeatures were rather different from those in vivo (12).Despite the gastric origin, this cell line showed somedifferentiation to squamous epithelial cells, but not toglandular cells. The general concept of differentiation fromcytotrophoblasts t@ syncytiotrophoblasts (7, 16, 22, 27) isbased primarily on the observations of several transitionalforms between these cell types in the norn@al as well asneoplastic tissues and on the autoradiographic findings ( I6).The present immunocytochemical stainings clearly demonstrated the presence of HCG in a small number of monoand possibly in multinucleated cells. The number of HCGsynthesizing and -storing cells correlated well with theamount of secretion into the medium. On the other hand, inthe cell line ultrastructurally typical syncytial cells wererare. Therefore, it can reasonably be assumed that a largenumber of cells with cytotrophoblastic features did notcontain any detectable amount of HCG, but the moredifferentiated mononuclear intermediate cells did containHCG and produce greater or lesser quantities of it. Thisassumption is supported by the ultrastructural finding ofintermediate cells possessing moderately developed endoplasmic reticulum and Golgi complexes, which are probablyessential for the synthesis, transport, and discharge ofsecretory proteins (23). Most investigators conducting invitroexperiments (9, 30) considered a cytotrophoblast as anHCG producer, because they thought that cytotrophoblastsalone were grown in culture. However, they provided noevidence that the colonies consisted exclusively of cytotrophoblasts. Also, they did not describe the intermediate celltype. The reason why syncytial cells were very few innumber in the cell culture can be explained by their rapiddegeneration and death under in vitro conditions. In ourpreliminary experiment on the nude mouse with heterotransplantation of the SCH cells,3 many HCG-positivemono- and multinuclear cells were found in tumors grownin the s.c. space, and intermediate and syncytiotrophoblastic cells were much more frequently encountered in vivothan in vitro conditions. Thus, it can be concluded that intermediate and syncytiotrophoblastic cells differentiatefrom cytotrophoblasts and become capable of synthesizingand storing HCG, as almost all immunohistochemists (15,20, 26) believe at the present.

It is well known that the alkaline phosphatase of placentalorigin is an organ-specific isoenzyme (28). Other workers,including one of the authors (10), demonstrated that theenzyme was localized on the free surface of syncytial cells ofthe chorionic villi. The presence of a substance almostidentical to HPALP on the free surface of a larger numberof SCH cells indicates that the cell line derived fromchoriocarcinoma maintains another characteristic of humantrophoblasts. The Regan isoenzyme, indistinguishable fromHPALP, as demonstrated by Fishman et al. (6), was foundin the sera of less than 10% of patients with cancer, andthere was some evidence that the source of the isoenzymewas the neoplastic tissue. In this connection, the SCH line

3 T. Kameya, Y. Shimosato, M. Tsumuraya, N. Ohsawa, and T.

Nomura, Human Gastric Choriocarcinoma Cell Line Serially Transplanted to Nude Mice, in preparation.

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may be a producer of Regan isoenzyme, either derived fromthe original gastric cancer and transferred to the line ornewly induced after the establishment of the line.

The fact that the source of the cell line was the stomachindicates dysdifferentiation from gastric mucosal cells totrophoblastic ones through gastric adenocarcinoma cells.The differentiation toward gastric cancer cells has not beenconfirmed in this in vitro investigation, although Oboshi eta!. (2 1) observed tubular structures in a tumor grown in ahamster's cheek pouch into which SCH cells were injected.

ACKNOWLEDGMENTS

Our thanks are extended to Dr. S. Oboshi who kindly supplied theoriginal cell line, to Dr. Y. Notake and Dr. Y. Shimosato for invaluableadvice and criticism, and to M. Hamano for technical assistance with tissueculture.

REFERENCES

I. Aono, T. Goldstein, D. P., Taymor, M. L., and Dolch, K. ARadioimmunoassay Method for Human Pituitary Luteinizing Hormone (LH) and Human Chorionic Gonadotropin (HCG) Using1251-Labeled LH. Am. J. Obstet. Gynecol., 98: 996- 1001, 1967.

2. Avrameas, S. Coupling of Enzymes to Proteins with Glutaraldehyde.Use of the Conjugates for the Detection of Antigens and Antibodies.Immunochemistry, 6: 43-- 52, 1969.

3. Baker, J. R. The Histochemical Recognition of Lipine. Quart. J.Microscop. Sci., 87: 441 -470, 1946.

4. Carter, J. E. Morphologic Evidence of Syncytial Formation from theCytotrophoblastic Cells. Obstet. Gynecol., 23: 647-656, 1964.

5. Evans, V. J., Earle, W. R., Sanford, K. K., Shannon, J. E., and Waltz,H. K. The Preparation and Handling of Replicate Tissue Cultures forQuantitative Studies. J. NatI. Cancer Inst., II: 907-927, 1951.

6. Fishman, W. H., Inglis, N. R., Green, S., Anstiss, C. L., Ghosh, N. K.,Reif, A. E., Rustigan, R., Kraut, M. J., and Stolbach, L. L.Immunology and Biochemistry of Regan Isoenzyme of AlkalinePhosphatase in Human Cancer. Nature, 219: 697-699, 1968.

7. Garancis, J. C., Pattillo, R. A., Hussa, R. 0., Schultz, J., andMattingly, R. F. Electron Microscopic and Biochemical Patterns of theNormal and Malignant Trophoblast. Am. J. Obstet. Gynecol., 108:1257 1268,1970.

8. Hamajima, Y., and Yasuda, K. Fluorescent Antibody Technique andEnzyme-labeled Antibody Technique, pp. 43-49. Tokyo: IgakuShoin, Ltd., 1971.

9. Jones, G. E. S., Gey, G. 0., and Gey, M. K. Hormone Production byPlacental Cells Maintained in Continuous Culture. Bull. Johns HopkinsHosp.,72:26 38,1943.

10. Kameya, T., Watanabe, K., Kobayashi, T., and Mukojima, T.Enzyme- and lmmuno-Histochemical Localization of Human Placental Alkaline Phosphatase. Acta Histochem. Cytochem., 6: 124-136,1973.

II. Kawaoi, A., and Nakane, P. K. An Improved Method of Conjugationof Peroxidase with Proteins. Federation Proc., 32: 840, 1973.

12. Knoth, M., Hesseldahl, H., and Larsen,J. F. Ultrastructure of HumanChoriocarcinoma. Acta Obstet. Gynecol. Scand.. 48: 100- 118, 1969.

13. Kohler, P. 0., and Bridson, W. E. Isolation of Hormone-Producing

Clonal Lines of Human Choriocarcinoma. J. Clin. Endocrinol. Metab.,32: 683-687, 1971.

14. Mayahara, H., Hirano, H., Saito, T., and Ogawa, K. The New LeadCitrate Method for the Ultrastructural Demonstration of Non-specificAlkaline Phosphatase (Orthophosphoric Monoester Phosphorylase).Histochemie, Ii: 88-96, 1967.

15. Midgley, A. R., and Pierce, G. B., Jr. Immunohistochemical Localization of Human Chorionic Gonadotropin. J. Exptl. Med., 115: 289-294,1962.

16. Midgley, A. R., Pierce, G. B., Jr., Deneau, G. A., and Gosling, J. R.G. Morphogenesis of Syncytiotrophoblast in vivo; angraphic Demonstration. Science, 14!: 349-350, 1963.

17. Midgley, A. R., Jr., Radioimmunoassay: A Method for HumanChorionic Gondadotropin and Human Luteinizing Hormone. Endocrinology,79:10-18,1966.

18. Morgan, C. R., Sorensen, R. L., and Lazarow, A. L. Further Studiesof an Inhibitor of the Two Antibody Immunoassay System. Diabetes,13:579-584,1964.

19. Nakane, P. K., and Pierce, G. B., Jr. Enzyme-labeled Antibodies forthe Light and Electron Microscopic Localization ofTissue Antigens. J.CellBiol.,33:307-318,1967.

20. Notake, Y., Yamaguchi, M., Nakagawa, K., and Uchiyama, N.Detection of HCG-producing Cells by Fluorescent Antibody Method(in Japanese). Clinical Gynecol. Obstet., Tokyo 19: 11-17, 1965.

21. Oboshi, S., Yoshida, K., Seido, T., Shimosato, Y., Koide, T., Sano,R., and Kitaoka, K. Culture of Primary Choriocarcinoma of Stomachand Its Gonadotropin Production in vitro. Proceedings of the 31stAnnual Meeting of the Japanese Cancer Association, No. I 15, p. 59,Tokyo: Japanese Cancer Assoc., 1972.

22. Okudaira, Y., Hashimoto, T., Hamanaka, N., and Yoshinaru, S.Electron Microscopic Study on the Trophoblastic Cell Column of

Human Placenta. J. Electron Microscopy Japan, 20: 93-106, 1971.23. Palade, G. E. Synthesis, Transport and Discharge of Secretory Pro

teins. In: T. Takeuchi, K. Ogawa, and S. Fujita (eds.), Histochemistry and Cytochemistry,p. 69. Kyoto,Japan:Societyof Histochemistry and Cytochemistry, 1972.

24. Pattillo, R. A., and Gey, G. 0. The Establishment of a Cell Line ofHormone-Synthesizing Trophoblastic Cells in vitro. Cancer Res., 28:1231-1236,1968.

25. Pattillo, R. A., Ruckert, A., Hussa, R. Bernstein, R., and Delfs, E.The Jar Cell Line. Continuous Human Multihormone Production andControls. In Vitro, 6: 398-399, 1971.

26. Pierce, G. B., Jr., and Midgley, A. R. The Origin and Function ofHuman Syncytiotrophoblastic Cells. Am. J. Pathol., 43: 153- 172,1963.

27. Pierce, G. B., Jr., Midgley, A. R., and Beals, T. F. An UltrastructuralStudy of Differentiation and Maturation of Trophoblast of theMonkey. Lab. Invest., 13: 451 -464, 1964.

28. Posen, S., Cornish, C. J., Home, M., and Saini, P. K. PlacentalAlkaline Phosphatase and Pregnancy. Ann. N. Y. Acad. Sci., 166:733@744,1969.

29. Rabson, A. S., Rosen, S. W., Tashjian, A. H., Jr., and Weintraub, B.Production of Human Chorionic Gonadotropin in vitro by a Cell LineDerived from a Carcinoma of the Lung. J. NatI. Cancer Inst., 50:669-674, 1973.

30. Stewart, H. L., Sano, M. E., and Montgomery, T. L. HormoneSecretion by Human Placenta Grown in Tissue Culture. J. Clin.Endocrinol., 8: 175-188, 1948.

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Fig. I. General view of the epithelial arrangement of the SCH cell line showing piling-up of cells, multinucleation, mitotic figures, and frequentvacuolated cells ( V). Giemsa staining, x 340.

Fig. 2. Ultrastructure of SCH cells: IC, intra- or intercytoplasmic vacuole; R, free ribosome; D, desmosome; T, tonofibrils; L, lipid vacuole. Uranylacetate and lead citrate staining, x 7,500.

Fig. 3. Ultrastructure of SCH cells with alkaline phosphatase activity (lead citrate method). M, microvilli; D, desmosome; T, tonofibrils; IC, intraand intercytoplasmic vacuoles. Uranyl acetate and lead citrate staining, x 12,000.

Fig. 4. Ultrastructure of SCH cells with an isolated desmosome. M, microvilli; L, lipid vacuole: G, Golgi complex; D, isolated desmosome. Arrowsindicate localization of alkaline phosphatase. Uranyl acetate and lead citrate staining, x 20,000.

Fig. 5. Ultrastructure of SCH cells with well-developed, dilated rough endoplasmic reticulum (RE). Uranyl acetate and lead citrate staining, x

7,500.Fig. 6. Amylase-sensitive PAS-positive coarsely granular material in the cytoplasm. x 680.Fig. 7. Glycogen (GI) particles aggregated in the cytoplasm and alkaline phosphatase activity on the plasma membrane (arrows). Uranyl acetate and

leadcitratestaining,x 27,000.

Fig. 8. Alkaline phosphatase activity (azo dye method). Black areas on the cell membrane were actually stained red in the original section. x 680.Fig. 9. Heat-stable (65°, 30 mm) alkaline phosphatase activity of SCH cells. x 680.Fig. 10. Complete inhibition of alkaline phosphatase activity by L-phenylalanine (0.05 M). Nuclei were stained with methyl green. x 680.Fig. II. Light microscopic localization of HPALP by peroxidase-labeled antibody method. Reaction deposits on the cell membrane appeared dark

brown in the original section. Nuclei staining with methyl green, x 680.Fig. 12. Electron microscopic localization of HPALP by peroxidase-labeled antibody method. Reaction deposits were localized on the cell surface

membrane, including microvilli (M). No metal staining, x 10,000.Fig. 13. Localization of HCG in SCH cells by FITC-labeled antibody method. x 1,200.

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1975;35:2025-2032. Cancer Res   Toru Kameya, Hiroyuki Kuramoto, Kenji Suzuki, et al.   ProductionChorionic Gonadotropin and Placental Alkaline Phosphatase A Human Gastric Choriocarcinoma Cell Line with Human

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