Coordinate Expression of Parietal Endodermal Functions in Hybrids of ...

Vol. 2, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1982, p. 331-337 0270-7306/82/030331-07$02.00/0

Coordinate Expression of Parietal Endodermal Functions in Hybrids of Embryonal Carcinoma and Endodermal Cells WILLIAM E. HOWEt AND ROBERT G. OSHIMAt* Department of Biology 16-627, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 21 June 1981/Accepted 20 October 1981

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9. Hybrid clones (ENEC1 to ENEC5) were isolated in HAT medium containing ouabain at a frequency of approximately 2 x 10-4. The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.

The stem cell of the teratocarcinoma is the embryonal carcinoma (ec) cell (17). Murine ec cells resemble pluripotent embryonic cells (21) and can arise spontaneously in certain strains of mice (42, 43) or from the transplantation of early mouse embryos (1 to 7 days of age) to ectopic sites (39, 41). Pluripotent ec cell lines retain the ability to differentiate to a variety of cell types in vivo and in vitro (10, 14, 22, 23). Other variant ec cell lines spontaneously differentiate very infrequently in vitro and form tumors composed nearly exclusively of ec cells (3, 13, 29, 30, 36, 40, 44). The fusion of two ec cell lines yields hybrids with ec characteristics, but variable development capabilities (30a, 33). We report here that the fusion of an ec cell line which spontaneously differentiates infrequently with a parietal endodermal cell line results in hybrids which express differentiated parietal endodermal characteristics and not embryonal carcinoma characteristics. MATERIALS AND METHODS Culture techniques and cell lines. Culture techniques have been described previously (30a). Dulbecco modified Eagle (DME) medium supplemented with pyruvate (110 mg/liter), glutamine (0.04%), penicillin (50 t Present address: La Jolla Cancer Research Foundation, La Jolla, CA 92037.

,Lg/ml), streptomycin (50 ,ug/ml), and 10% fetal bovine serum was used. Stock cultures were usually cultivated without antibiotic. The near-diploid FOT5 cell line was derived from F9.22 (30) and is ouabain and thioguanine resistant. FOT5 and F9.22 cells both form embryonal carcinoma tumors when injected into strain 129 mice and spontaneously differentiate very infrequently. in vitro (30a). PFHR9 is a near-diploid extra-embryonic parietal endodermal cell line and was derived from the ec cell line PCC4-F (4). All cell lines were derived from strain 129 mice. Cell fusion. Mixtures of FOT5 and PFHR9 cells were fused by the polyethylene glycol (PEG, molecular weight, 6,000; Sigma Chemical Co., St. Louis, Mo.) procedure (28). Confluent mixed cultures were exposed to 50%o PEG (wt./vol.) in DME medium containing 10% dimethylsulfoxide (DMSO) for 1 min, washed six times with medium containing 10%o DMSO, and washed once with DME containing 10o fetal bovine serum. The following day the cells were dissociated and filtered through 15-1Lm mesh-nylon cloth to remove aggregates. Between 104 and 106 cells were seeded in DME containing 10% fetal bovine serum, 2 mM ouabain, 10 FLM hypoxanthine, 400 nM aminopterin, and 16 ~iM thymidine (HAT medium) (18). After 2 weeks, representative plates were fixed in 3.7% formaldehyde in phosphate-buffered saline, washed twice in methanol, and stained with Giemsa stain for morphological examination and colony counting. Hybrids were picked by using glass cloning cylinders, expanded in selective medium, and recloned to elimi-

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nate possible contaminants due to metabolic cooperation (7). Each original hybrid was picked from an independent dish in which 104 cells had been seeded. Analyses of these hybrids (designated ENEC1 through ENEC5) were performed between passages 5 and 15 after recloning. ENEC1 and ENEC4 cell lines were isolated from a fusion in which the PFHR9 cells were in threefold excess of FOT5 cells. ENEC2, ENEC3, and ENEC5 were isolated from a fusion in which FOT5 cells were in 10-fold excess over PFHR9 cells. DNA determinations. Single-cell DNA contents were measured by flow cytofluorometry after staining with mithramycin as previously described (8, 31). The instrument used for the DNA determination was an ICP-22 (Ortho Diagnostic Systems, Westwood, Mass.) (45). This is a flow system that uses a mercury arc lamp, optically filtered to 380 nm, as the excitation source. The fluorescence emitted from the stained cells is filtered and transmitted to two photomultiplier tubes (PMT-1 and PMT-2). PMT-1 (540 to 1,200 nm) and PMT-2 (470 to 510 nm) quantitate the fluorescence from the cells, and the PMT output is routed to the signal-processing system of the Ortho System 50 cell sorter. The signals were converted to numerical values on the basis of analog signal peak heights by using a 120-ps sample interval (slow mode). Fluorescent 1/4and 1/2-bright, 10-,um-diameter spheres (Coulter Electronics, Woburn, Mass.), with a coefficient of variation of 3.3 and 2.7%, respectively, were used to optimize and standardize the system. Once the gains were standardized, they remained constant and were monitored by using the spleen cell standard or PFHR9 parent cells as a reference. Metaphase chromosomes were prepared by standard procedure. Cells were treated for 2 to 3 h with 10 ng of colchicine per ml, dissociated with 0.25% trypsin, incubated at 37°C in hypotonic medium for 10 min, and fixed in methanol-acetic acid (30:12). Slides were prepared, air dried, and stained with Giemsa stain. Embryonal carcinoma characteristics. The tumorgenicity of each parent and hybrid cell line was tested by injecting two male mice (strain 129) each with 2 x 106 to 6 x 106 cells subcutaneously (30a). In addition, six mice were each injected with up to 3 x 107 ENEC1 cells. If the mice produced no tumor, they were observed for 5 to 6 months before they were sacrificed. Tumors were fixed in formaldehyde in phosphate-buffered saline, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Indirect immunofluorescence staining for a stagespecific embryonic antigen (SSEA-1) was performed as described by Solter and Knowles (37). Alkaline phosphatase activity was measured by the procedure of Bernstine et al. (3). Parietal endodermal characteristics. Indirect immunofluorescence staining for the basement membrane protein laminin (46) was performed as previously described (30), except that cells were first fixed in 3.7% formaldehyde in phosphate-buffered saline for 5 min at room temperature before treatment with cold methanol. The basement membrane protein antiserum was prepared by immunizing a rabbit with the basement membrane glycoproteins secreted by the PFHR9 cell line by the procedure of Chung et al. (5). The specificity of the serum was tested both by immunofluorescence and immunoprecipitation on a variety of cell

MOL. CELL. BIOL.

lines and found to be identical with the GP2 basement membrane antiserum (30). Photographs (1 to 5 s) were taken with an Olympus OM2 camera attached to a Dialux 20 E. B. Leitz fluorescent microscope. Kodak Tri-X film was developed for 20 min at room temperature in Microdol-X. Plasminogen activator activity of single-cell suspensions was measured as previously described (30). Immunoprecipitation of [35S]methionine-labeled endo A and endo B cytoskeletal proteins was performed as previously described (29a), except that the final concentrations of sodium dodecyl sulfate and Nonidet P40 detergents in the cell lysates were increased to 0.5 and 1%, respectively. RESULTS

Hybrids of endodermal and embryonal carcinoma cells (ENEC1 through ENEC5) were selected from PEG-fused mixtures of the PFHR9 and FOT5 cell lines in HAT medium (18) containing 2 mM ouabain at a frequency of approximately 2 x 10-4. All the primary presumptive hybrid clones (1,358) had endodermal morphology. A control fusion with 107 PFHR9 parental cells alone did not yield any progressively growing colonies in selective medium. In addition, no ouabain-resistant PFHR9 variants were found among 2 x 107 cells screened. The spontaneous reversion of FOT5 cells to HAT resistance is less than 2 x 10-6 (30a). These results indicate that all of the ENEC presumptive hybrids probably were derived from the fusion of PFHR9 and FOT5 cells. This conclusion is supported by the results of measurements of the single-cell DNA contents of the hybrid and parental cells (Table 1). The amount of DNA in the ENEC hybrids varied from 3.7 to 4.4 C (spleen cell standard, 2.0 C). No evidence was observed in the flow microfluorometer profiles for cells containing the 2.1-C parental DNA contents. The average chromosome number of each hybrid was approximately the sum of the two parental lines (Table 1). The FOT5 cell line has one to two metacentric chromosomes. In contrast, the PFHR9 cell line has three metacentric chromosomes. Four of the five ENEC hybrids contained the number of metacentric chromosomes expected for cells derived from the fusion of one PFHR9 cell with one FOT5 cell. The remaining hybrid, ENEC5, contained an extra metacentric chromosome (Table 2). However, because the average total chromosome number and the single-cell DNA contents of the ENEC5 cell line are consistant with an origin from the fusion of only one embryonal carcinoma and one endodermal cell, it appears likely that the extra metacentric chromosome may have been formed by the fusion of two acrocentric chromosomes during the propagation of the hybrid cell population. Expression of embryonal carcinoma character-

ENDODERMAL-EMBRYONAL CARCINOMA HYBRIDS

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TABLE 1. The DNA contents and chromosome number Range"

No. of metacentric chromosomes per metaphase

38-41 37-41

1.4 3.0

Chromosome no. Cells

Control Spleen cells Parents FOT5-ec

PFHR9-endodermal

DNA contents

Meana

2.0 ± 0.1

40

2.1 ± 0.2 2.1 ± 0.2

39 39

Hybrid 4-5 75-82 78 4.2 ± 0.4 Expected 4.7 67-78 72 4.4 ± 0.4 ENEC1 4.2 72 64-79 3.8 ± 0.6 ENEC2 5.0 70 65-78 4.0 ± 0.7 ENEC3 4.7 62-77 69 3.7 ± 0.4 ENEC4 6.0 64-78 73 4.2 ± 0.6 ENEC5 a Fifteen well-spread, non-broken metaphases were counted. b Some metaphases had higher chromosome number. Of the FOT5 and PFHR9 chromosome preparations 1 and 8%, respectively, had more than 50 chromosomes. Of the hybrid metaphases, 5 to 10% contained more than 90 chromosomes. c Coefficient of variation.

istics. Embryonal carcinoma cells are highly tumorigenic when injected into syngenic hosts. The FOT5 ec cell line, like its parental line, F9 (3), forms embryonal carcinoma tumors 1 to 2 cm in diameter 1 to 2 weeks after the subcutaneous injection of 2 x 106 cells. In contrast, PFHR9 does not form tumors in strain 129 mice even after the injection of as many as 3 x 107 cells. Each ENEC hybrid was tested by the injection of between 2 x 106 and 3 x 107 cells. No tumors developed in 16 mice within the 5-to6-month observation period. The FOT5 cell line, like other embryonal carcinoma cell lines, expresses a stage specific embryonic antigen (SSEA-1) that is also found in early mouse embryonic cells (11, 37). A mono-

clonal antibody against SSEA-1 was used to test the parental and hybrid cell lines for the presence of the antigen by indirect immunofluorescence. The results are shown in Table 2. Neither the PFHR9 endodermal parental line nor any of the ENEC hybrids expressed SSEA-1. Embryonal carcinoma cells have relatively high alkaline phosphatase activity (2, 3, 48), whereas the PFHR9 cell line has little of this enzyme (4). The alkaline phosphatase activities of the ENEC hybrids were approximately the same as that of the PFHR9 endodermal cell line (Table 2). These results were confirmed with several independently prepared lysates. Expression of parietal endodermal characteristics. PFHR9 cells synthesize and secrete plas-

TABLE 2. Characteristics of the parents and hybrid cells Alkaline

Cells

Parents FOT5-ec PFHR9-endodermal

SSEA-ve(% Positive cells) 98