Regulation of phenotype in somatic cell hybrids derived by fusion of teratocarcinoma cell lines with normal or tumor-derived mouse cells

DEVELOPMENTAL

BIOLOGY

Regulation

81,

245-254 (1981)

of Phenotype in Somatic Cell Hybrids Derived by Fusion of Teratocarcinoma Cell Lines with Normal or Tumor-Derived Mouse Cells RUDOLF GM~~R,’ BARBARA The Wistar Institute

B. KNOWLES, AND DAVOR SOLTER

of Anatomy and Biology, Philadelphia,

Received May 23, 1980; accepted in revised form

Pennsylvania

19104

July 7, 1980

The phenotypes of somatic cell hybrids between murine embryonal carcinoma cell lines, F9 BrdU 7C12 and PCC4 aza 1, and normal murine splenic lymphocytes or thymoma-derived cell lines were compared. Analysis of morphology in vivo and in vitro of cell surface markers and of the karyotype of these cloned hybrid cells did not reveal any simple mechanism for the regulation of the phenotype of such hybrids. Hybrids of either the embryonal carcinoma cell phenotype or of a differentiated morphology (resembling neither parental cell) but not of lymphoid morphology can be derived from fusions of this type. Moreover, transition from one phenotype to the other (ECC -+ differentiated cell and differentiated cell - ECC) can be found with passage of clonally derived hybrid cell lines. Coordinate control of the phenotypic markers of the state of differentiation in these hybrid cells was found. INTRODUCTION

tained the morphological and biochemical features of ECCs. These observations led to two very interesting Most somatic cell hybrids between “undifferentiated” conclusions: (1) ECCs must have regulative mechanisms and differentiated cells do not express the traits charable to extinguish expression of traits of differentiated acteristic of the differentiated parental cell (for review parental cells; (2) although equipped with mechanisms see: Ephrussi, 1972; Davis and Adelberg, 1973; Davidson, 1974; Ringertz and Savage, 1976). Extinction of that enable them to stabilize their differentiated state, differentiated functions is probably due to the sup- the differentiated parental cells are not able to restrict pressive activity of regulatory substances from the a pluripotent genome to their own cell lineage. Similar “undifferentiated” parental cell (Schneider and Weiss, results were reported by Andrews and Goodfellow 1971; Croce et al., 1973; Ringertz and Savage, 1976), since (1980). However, McBurney (1976, 1977) obtained hyprolonged cultivation of hybrid cell lines can lead to the brids from fusions of two C3H-derived ECC lines with reexpression of differentiated traits, apparently as a Friend leukemia cells that grew in suspension and that consequence of chromosome loss (Klebe et aZ., 1970; were able to produce hemoglobin chains in the presence Weiss and Chaplain, 1971). Embryonal carcinoma cells of inducers, from both ECC and Friend cell-derived (ECCs), the undifferentiated, pluripotent stem cells of genes. In this case the differentiated cells can “domiteratocarcinomas are the ideal undifferentiated fusion nate” the ECC phenotype. Furthermore, the induction partner for this type of study. The pluripotency of these of the synthesis of a cell lineage-specific marker protein cells has been proven both by the formation of tera- (hemoglobin) from ECC genes indicates that the diftocarcinoma, containing well-differentiated tissues, ferentiated cell can restrict the genome and regulation from a single injected ECC (Kleinsmith and Pierce, systems of the ECC to its own cell lineage. Other fusions 1964), and by formation of chimeric mice from ECC between ECC and a number of different transformed injection into mouse blastocysts (Brinster, 1974; Pa- cell types, also resulted in hybrids phenotypically resembling either the differentiated parent or neither paioannou et al., 1975; Mintz and Illmensee, 1975). Somatic cell hybrids between ECCs and a variety of parental cell (Finch and Ephrussi, 1967; Jami et al., differentiated cell types have been produced, and the 1973; McBurney, 1976; Evans, 1975; Bernstine et aZ., control of phenotypic expression in such hybrids ana- 1977; Watanabe et al., 1978; Litwak and Croce, 1979). lyzed. Miller and Ruddle (1976, 1977a,b) fused cells of In light of such dissimilar regulation of phenotypic the ECC line PCC4 aza 1 with either mouse thymocytes expression in ECC hybrids with differentiated cells, we or Friend leukemia cells and obtained hybrids that re- decided to hybridize two different ECC lines with both i Present address: University of Zurich, Immunobiology Research normal and tumor-derived lymphoid cells. From each Group, Scbonleinstrasse 22, CH-8032 Zurich, Switzerland. fusion, regardless of the parental cells, hybrid clones 245

0012-1606/81/020245-10$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

246

DEVELOPMENTAL BIOLOGY

were derived with either the undifferentiated (ECC) or a fibroblastoid phenotype. No hybrids resembling the lymphoid parental cells were obtained. Hybrid clones with the ECC morphology can subsequently differentiate in vitro. On the other hand, the fibroblastoid hybrid clones can, on passage in vitro, give rise to cells with the ECC morphology. We propose two possible mechanisms to account for this transition, one of them regulatory (differentiation of ECC) and the other, specific chromosome change. MATERIALS

AND METHODS

Cell lines and culture conditions. Two teratocarcinoma stem cell lines were used for fusions: F9 BrdU 7C12 (Gmiir et al., 1980), a nullipotent ECC, and PCC4 aza 1 (Nicolas et ah, 1976), a pluripotent ECC. Both cell lines are derived from the 129/Sv mouse (H-2bc) and are sensitive to HAT-selective medium (Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 13.6 pg/ml hypoxanthine, 0.025 pg/ml methotrexate, 3.9 pg/ ml thymidine, and 0.02 pg/ml glycine). The BW5147 cell line, derived from an AKR/J (H-zk) T-cell lymphoma (resistant to 10 /*g/ml 8-azaguanine) (Hyman and Stallings, 1978), and the GF-7 cell line, derived from the L5178Y (H-zd) T-cell lymphoma, resistant to 30 pg/ml bromodeoxyuridine (BrdU) (Stadler and Adelberg, 1972), are also both killed in HAT-selection medium. The three cell lines K129SV (H-2b) KC3HSV (H-zk) and KFISV (H-2d,b) are SV40-transformed kidney-derived fibroblasts (Knowles et ab, 1979). Cell hybridization. Splenocytes were prepared by gentle teasing of freshly dissected C3H/HEJ (H-2k) or CBA/J (H-2k) spleens as previously described (Gmiir et aZ., 1980). PCC4 aza 1 and F9 BrdU 7C12 were harvested with 0.25% trypsin in Dulbecco’s modified phosphate-buffered saline (PBS), calcium and magnesium free, containing 0.1% EDTA (ethylenediaminetetraacetic acid). Single cell suspensions of BW5147 and GF7 were prepared by vigorous pipetting in culture medium. The two cell suspensions to be fused were mixed, washed once in serum-free medium, and the cell pellet was resuspended in 45% polyethylene glycol (PEG) 1000 (Galfre et ah, 1977; Gmiir et ah, 1980). Cells were seeded in 24-well tissue plates (FB16-24TC, Linbro Chemical Co.) in HAT-selective medium. Hybrid clones were isolated from independent wells containing a single colony and subclones were obtained by the limiting dilution technique. Hybrid clones were obtained from the following fusions: 1. PCC4 aza 1 X C3H splenocytes (477 series) 2. PCC4 aza 1 X CBA splenocytes (52 series)

VOLUME 81, 1981

3. PCC4 aza 1 X GF-7 (72 series) 4. F9 BrdU 7C12 X C3H splenocytes (67 series) 5. F9 BrdU 7C12 X BW5147 (978 series)

Isoxyme and chromosome analysis. Electrophoresis of extracts of hybrid cell lines was carried out on horizontal 12% starch gels. Glucose phosphate isomerase (GPl, EC 5.3.1.9) and isocitrate dehydrogenase (IDH, EC 1.1.1.42) were visualized by standard techniques (Nichols and Ruddle, 1973). Metaphase spreads of exponentially growing cells were prepared, stained with Giemsa, and intact metaphases were photographed and analyzed. Antisera. Ascites fluid from mice injected with B22249 Rl hybridoma cells (Lemke et ah, 1979) was the source of the monoclonal antibody (IgGza class) recognizing the H-2.2 private specificity (H-2Db), and was used at a 1:4000 dilution in indirect-antibody binding radioimmunoassay (RIA) and a 1:lOO dilution for fluorescent assays. Ascites fluid from a BALB/c mouse, injected with 11-4.1 hybridoma cells (Oi et ah, 1978) was the source of monoclonal antibody (IgGza) reacting to a public specificity common to H-2Kk, H-2KP, H-2Kq, and H-2K’ molecules and was used at a 1:lOOO dilution in RIA. Cell culture fluid from hybridoma MN-3706 secreting antibody against H-2Kd was obtained from Dr. Markus Nabholtz, ISREC, Epalinges, Switzerland, and was used undiluted. The monoclonal antibody (IgM) that detects a stagespecific embryonic surface antigen (SSEA-1) expressed on early embryo and ECC has been described in detail (Solter and Knowles, 1978). The anti-SSEA-1 reagent used was ascites fluid from a tumor-bearing BALB/c mouse and was used at a dilution of 1:32,000 in RIA and 1:lOO in fluorescent assays. Monoclonal antibodies against Thy-l.1 and Thy-l.2 (New England Nuclear) as well as conventional antiThy-l.1 and anti-Thy-l.2 sera (Bionetics and Searle Diagnostic, respectively) were used in RIA or absorption analysis, respectively. Preparation of cells and immunoassays. Tissue culture cells were harvested, counted, and suspended in 0.01 A! N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes)-buffered (Sigma) MEM containing 10% FBS. Thymocytes were prepared from thymus of young mice by teasing and centrifugation through a Ficoll-Hypaque (Lymphoprep, 1.077 pg/ml, Nyegaard and Co., Oslo, Norway). For all incubations of target cells with antisera Linbro IS-RB-96 round-bottom microtiter plates were used. The RIA and preparation of lz51 rabbit anti-mouse IgM and IgG are described elsewhere (Aden and Knowles, 1976). Results are reported as the average counts per minute (cpm) from triplicate samples minus

GM~R, KNOWLES, AND SOLTER

247

Phenotype of Teratocarcinoma H&rids

FIG. 1. Starch gel electrophoresis of hybrid clones. (a) Glucose phosphate isomerase in hybrid and control cells. Lane (1) C3H/HeJ spleens; (2) 477-l GG; (3) 477-l P; (4) 477-l I; (5) 477-l B; (6) PCC4 aza 1; (7) C3WHe.I and PCC4 aza 1 mixed 1:l prior to extraction; (8) 477-l DD. The three dimeric isozymes formed by the subunits A and B are labeled AA, AB, and BB. (b) Isocitrate dehydrogenase in hybrid and control cells. Lane (1) F9 BrdU 7C12; (2) 978 cl 1-5; (3) 978 cl l-6; (4) 978 cl 2-l; (5) BW5147.

the average responding

cpm bound to cells incubated with a corof control myeloma-induced ascites

dilution

fluid. 976tF9

I BW5147)

?2(PCC4

I GF71

I

n 4

67tF9

x

x C3H)

62(PCC4

I CBA1

477cPCC4

I C3i-i)

For immunofluoresence tests, cells were incubated with monoclonal reagents followed by a second incubation with 1:lO diluted fluorescein isothiocyanate-labeled goat anti-mouse IgGz serum (GAM IgGz-FITC) from Meloy Laboratories, or tetramethylrhodamine-labeled goat IgG anti-mouse IgM (GAM IgM-TMR; heavy chain specific) from Cappel Laboratories. Cells were examined using a Leitz Orthoplan microscope equipped with fluorescent epiillumination, a 50X water immersion lens and appropriate filters. Tumorigenicity. Parental and hybrid cells were harvested, resuspended at 10’ cells per milliliter PBS, and 0.5 ml of cell suspension was injected subcutaneously into F1 mice. Mice were inspected weekly for tumor growth. When tumors reached approximately 1 cm in diameter, the animals were sacrificed and tumors examined histologically. TABLE 1 EXPRESSION OF SSEA-1 AND H-2Db

CONTROLS 31

FIG. 2. H-2 and SSEA-1 expression on hybrid and control cells-as detected by indirect antibody binding radioimmunoassay with monoclonal antibodies. Data are expressed as average counts per minute (cpm) bound to triplicate cell samples (1 X 10’) minus the cpm bound to triplicate samples of each cell (200-400 cpm) when incubated with appropriately diluted cell culture supernatant or from ascites fluid from P3X63 Ag8 tumors. The input was 50,000 cpm as rabbit IgG anti-mouse IgG (heavy and light chain specific).

Percentage positive cells” Cells

H-2Db

SSEA-1

F9 BrdU 7C12 BW 5147 K129SV 978-l-5 D8 978-l-5 H9 978-l-5 H12

0 0 63 86 79 75

93 0 0 0 30 5

“Cells stained with monoclonal anti-H-2Db and anti-SSEA-1 reagents as first reagents and with GAM IgGz-FITC and GAM IgMTRM as second reagents.

248

DEVELOPMENTALBIOLOGY RESULTS

Hybrid Nature and Antigen Expression of Isolated Clones We used several criteria to determine if clones isolated from these different fusions were hybrids. The clones were isolated as substrate-attached cells in HAT medium; no suspension cells were observed. The mean chromosome number of each clone exceeded that of the parental cells and the clones usually contained the marker chromosomes from the parental cells. All clones had hybrid isozyme patterns: the parental cells of the 67, 52, and 477 series hybrid clones contained differing alleles for GPI, whereas parental cells of the 978 and 72 series hybrid clones have differing alleles for IDH. Both homodimeric and heterodimeric forms of these enzymes were found in the hybrids (Figs. la and b). An additional marker for the hybrid nature of the isolated clones would be the expression of the allelic antigenic determinants of the H-2K and D molecules from each of the parental cells. However, expression of H-2 mol-

VOLLJMES~,~~~~

ecules is a differentiated function with respect to the ECC phenotype so that hybrids resembling ECC would not be expected to express H-2 (Artzt and Jacob, 1974; Forman and Vitteta, 1975; Gmtir et al, 1980). Furthermore, in teratocarcinoma-derived hybrids not of the ECC morphology, the H-2K and H-2D coded molecules can be independently expressed (Gmi.ir et al., 1980). Thus by the criterion of codominant expression of both H-2 alleles, only some clones can be shown to be hybrid. Using a combination of isozyme, chromosome, and H2 analysis each of the clones and subclones have proven hybrid. Expression of Cell Surface Markers To characterize the differentiated state of the hybrid clones we examined the presence or absence of three independent surface antigens, appearing at different stages of embryonic development: stage-specific embryonic antigen (SSEA-l), expressed on ECC and not on differentiated mouse cell lines in culture (Solter and

FIG. 3. Morphology of parental and hybrid cells in culture. (A) F9 BrdU 7C12; (B) BW5147; (C) hybrid clone 978-l-5; (D) hybrid clone 9’78l-6. X200.

GM~~R,KNOWLES,ANDSOLTER

Phenotype

Knowles, 1978); H-2, expressed on most murine somatic cells and not ECC; and Thy-l, an antigen expressed on murine T cells. The majority of the hybrid clones express either H-2 or SSEA-1 although some clones or subclones express both (Fig. 2, Table 1). Microscopic examination of such clones (67-2-1, 67-2-5, 978-l-5 H9) shows cells of two distinct morphologies, EC and fibroblastoid, in the culture. Detailed analysis of H-2 expression of these hybrids is presented elsewhere (Gmtir et al., 1980). Neither the Thy-l.1 nor the Thy-l.2 gene products are expressed on any of the hybrid clones obtained (data not shown). The parental cells of both the 978 and 72 hybrid series are derived from thymomas and express the Thy-l.1 (BW5147) or Thy-l.2 (GF-7) antigen. In the case of the 978 hybrids, the presence of at least one chromosome 9, which contains the genes for both Thy-l and malic enzyme (MOD-l), from each parental cell is indicated by isoenzyme analysis (data not shown).

of Te-ratocarcinoma

Hybrids

249

Morphology of Hybrids and Changes with Culture Hybrid clones from the 978 fusion were fibroblastoid upon isolation. In contrast to the parental lines (Figs. 3A and B), cells of these hybrid clones (Figs. 3C and D) are flat, large, and elongated, bearing none of the distinguishing morphologic criteria of either parental cell. Forty passages following subcloning of the 978-l-5 hybrid ECC-like islands were noted in several subclones (representative 978-l-5 H9). This morphologic observation was substantiated by the detection of a subpopulation of SEA-1 positive cells in the culture (Table 1). Unlike the 978 hybrid clones, the hybrids derived from fusion with normal splenocytes (67, 52, and 477) resembled the teratocarcinoma parent on isolation. Detailed morphologic investigation of the 477 series illustrates this point. The PCC4 aza 1 parental cell line (Fig. 4A) does not routinely differentiate in culture.

FIG. 4. Morphology of parental and hybrid cells in culture during the exponential growth phase and after “aging.” (A) PCC4 aza 1; (B) 1477days after last subculturing. Note cell differentiation along the border of the ECC-like cc)lony 1DD; (C) 477-l GG; (D) 477-l GG “aged”-21 in 477 -1 GG (C). x200.

250

DEVELOPMENTALBIOLOGY

Examination of two hybrid clones 3 days after trypsini&ion shows groups of cells with ECC morphology comprising most of the culture (Figs. 4B and C). However, a small proportion of cells at the borders of these ECClike colonies are of a different morphology (Fig. 4C). If these EC&like clones are refed on a biweekly schedule for up to 3 weeks following subculture, the majority of the cells in the culture differentiate (Fig. 4D). These morphologic changes are paralleled by changes in the antigen expression in the culture. Cells from cultures tested 3 to 4 days postsubculturing were H-2 negative and SSEA-1 positive (Fig. 2); however, the same clones tested 21 days postsubculturing were H-2 positive (Gmiir et aZ.,1980). By passage 25 after isolation of the 67 hybrid clones, two morphologically distinct populations were seen in the cultures. Four subclones were isolated from the 67-2-5 hybrid clone; two morphologically ECC (subclone E4, F5) and two with large, flat epithelioid cells (subclone D6, D12). All four subclones are proven hybrid cells; the two ECC-like clones express both parental isozymes of GPI and the heteropolymer

VOLUME81. 1981

and the two differentiated clones express determinants of the H-2Db and Kk molecules (Fig. 2). The 72 series hybrid clones were initially isolated as ECC. As the hybrids were passaged, flat epithelioid cells were observed in most of the hybrid clones. This is reflected in the antigenic profile (Fig. 2). Chromosomal Composition of Hybrid Cells A detailed investigation of the chromosome content of the 978 hybrid clones and subclones throughout approximately 1 year of their passage history was conducted. These hybrid clones appear to derive from fusion of one F9 with one BW5178 cell with gradual reduction of the average number of chromosomes on passage although the chromosome number is relatively stable (Table 2). Each of the 477-l hybrid clones appears to be the result of fusion of one PCC4 cell with one C3H spleen cell (Table 3). The overall chromosome number of the 477 hybrids is lower than that in the 978 hybrids, how-

TABLE 2 CHROMOSOMAL CONSTITUTIONOF 978 SERIESHYBRID CELLS

Cell line F9 BrdU 7C12 BW 5147 Hybrid expected 1:l 978-l-5

978-l-6

978-2-l 978-l-5 D8 978-l-5 Dll 978-l-5 H9 978-l-5 H12 978-2-l 2B6 978-2-l 2F8

Passage No.

No. of chromosomes*

7 32 66 5 32 62 32 65 12 45 12 45 12 45 45 10 10

40 43 83 77 72 68 73 69 6’7 75 67 77 77 74 69 75 75 67 65 65

Range

No. of metaphases examined

39-42 40-45

35 26

69-81 66-79 65-71 71-75 65-75 65-71 69-80 63-74 73-82 73-81 69-78 67-72 70-79 71-83 63-72 63-66 62-67

19 35 14 17 20 16 27 15 39 17 18 13 11 16 17 11 12

Marker chromosomes I”

IId

IIId

1.0” 0 1 0.94

0 1.0 1 0.94

0 1.0 1 0.33

0

1.0

0

0.67

0.89

0.11

a Number of passages after fusion (normally one passage per week) or subcloning. * Average chromosome number. ’ Marker chromosome of F9 BrdU 7C12. dMarker chromosomes of BW 5147. ’ Ratio of number of metaphase spreads containing given marker/total number of spreads examined for presence of markers.

GM~~R,KNOWLES,ANDSOLTER

Phenotype

ever, the proportion retained (of the total chromosome number given a 1:l fusion) is approximately the same. Chromosomes of subclones of the 67-2-5 hybrid were analyzed to find if the transition between the ECC phenotype and fibroblastoid morphology was the result of obvious chromosome change (Table 3). The two subclones of the ECC phenotype each contained the F9derived marker chromosome but only 80-90% of the number of chromosomes expected for a 1:l fusion. In the fibroblastoid clones, extensive chromosome rearrangement was seen. Although the average chromosome number of the 67-2-5 D12 subclone paralleled that of the ECC-like subclones, new metacentric chromosomes, morphologically different from the submetacentric F9 marker chromosome and multiple minute chromosomes were found. The 67-2-5 subclone D6 contained these multiple metacentric and minute chromosomes and approximately double the chromosome number of the D12 clone (Table 3). Tumorigenicity

of Hybrids

To determine if the morphologic and antigenic phenotypes of the hybrid clones were sufficiently stringent hallmarks of the teratocarcinoma phenotype, these hybrids were injected into syngeneie F1 hybrid mice. Of the 28 (C3H/HeJ X 129/S) F1 mice injected with the 978 series hybrids only 11 developed tumors (Table 4). TABLE

251

of Teratocarcinoma Hybrids

These tumors resembled neither the teratocarcinoma nor the thymoma parental tumors (Fig. 5D); they were undifferentiated sarcomas (Fig. 5C). On the other hand the 477 hybrid clones gave rise to tumors histologically indistinguishable from undifferentiated teratocarcinomas (Fig. 5A) or differentiating teratocarcinomas (Fig. 5B). Injection of the 52 series hybrid clones and 67 clone 2-l resulted in formation of tumors composed predominantly of ECC and several derivatives. Tumors arising from injection of 67 clone 2-5 were undifferentiated sarcomas. The 67 clone 2-5 subclones D6 and D12 gave rise to undifferentiated sarcomas and the subclones E4 and F5 to teratocarcinomas. DISCUSSION

We feel that no definitive rules can be discerned as to the regulation of the parental phenotypes in somatic cell hybrids between ECCs and normal or tumor-derived differentiated cells. As to the phenotype of such hybrids, three possibilities exist. First, the hybrids can retain the ECC phenotype. This has been reported in hybrids between ECCs and normal thymocytes (Miller and Ruddle, 1976; Andrews and Goodfellow, 1980; Rousset et al., 1980), spleen cells (Gmiir et al., 1980; and in hybrid clones of the 477, 52, and 67 series described in this manuscript), Friend leukemia cells (Miller and Ruddle, 1977a,b), and thymoma-derived cell lines (Rous3

CHROMOSOMALCONSTITUTIONOF~T~ ANDRE SERIESHYBRIDCELLS No. of Cell line PCC4 aza 1 Spleen cells Hybrid expected 1:l 477-l B 477-l I 477-l P 477-l DD 477-l GG

F9 BrdU 7C12 Spleen cells Hybrid expected 1:l 67-2-5 F5 67-2-5 E4 67-2-5 D12 67-2-5 D6

Passage no.

No. of chromosomes

33 7 36 32 34 26

14-26 26 28 24

a Average chromosome number. b Ratio of the number of metaphase spreads containing ’ Minute chromosomes also present.

39” 40 79 65 71 67 55 64 67

40 40 80 68 65 67 124

the marker/total

Range 37-40 59-71 63-79 63-71 52-61 56-70 62-71

39-42 64-72 64-66 64-70 120-130

number of

Marker

metaphases counted 16 21 22 25 25 37 32

35 27 18 21 32

chromosomes

I

II

l.Ob

0.92 0 1 0 0.52 0.42 0.60 0.57 0.25

0 1 1.0 1.0 1.0 0 0.91 0.97

1 0 1 1 1 1

Additional metacentric l-3 1 5-7c

1

10-13"

metaphasespreadsexaminedfor presenceof markers.

252

DEVELOPMENTAL BIOLOGY

VOLUME

81, 1981

the hybrids can phenotypically resemble the differentiated parent. Such a phenotype has been described in all of the fusions in which the parental cell line is fiNo. of mice with broblastic (Finch and Ephrussi, 1967; Jami et al., 1973; tumors/total First time Time period McBurney, 1976; McBurney and Strutt, 1979; Rousset number of mice tumor growth of Injected cells injected noticed” observation” et ah, 1979; Andrews and Goodfellow, 1980). Several different teratocarcinoma cell lines have been used as F9 BrdU ‘7C12 4/4 7 15-30 fusion partners with two different fibroblastic cell lines BW 5147 7 23 4/4 (L cells and 3T3). The differentiated phenotype has also 978-l-5 220 o/4 been observed in one instance of fusion between ECC 220 978-l-6 o/2 978-2-l 18 42 5/5 and Friend erythroleukemia cells (McBurney, 1977; 978-l-5 Dllb 49 88 2/4 McBurney et aZ., 1978). None of the hybrid clones re978-l-5 H12b 170 o/5 ported herein resemble the differentiated parent. Third, 978-l-6 C5” 28 86 4/8 the hybrids may not resemble either of the parental cells as has been described in ECC fusion with thya Days after injection. ’ Tested 2 and 13 passages after subcloning (subcloning at passage momas (series 978 and 72 hybrid clones presented 15 of 978-l-5). herein), with a rat hepatoma (Litwak and Croce, 1979) ‘Tested 5,24, and 26 passages after subcloning (subcloning at pasand with a plasmacytoma (Correani and Croce, 1980). sage 5 of 978-l-6). Several different ECC lines were used to obtain these results. From this brief review, it becomes apparent then that set et al., 1980, as well as the ‘72series reported herein). Two different ECC lines, albeit derived from the same no rule about the phenotype or the regulation of pheoriginal tumor, were used in all of these fusions. Second, notype of an ECC fusion can be made. This point is TABLE 4 TUMORIGENICITY OF HYBRID AND PARENTAL CELLS

FIG. 5. Histology of tumors formed by injection of hybrid cells. (A) Undifferentiated embryonal carcinoma from 477-l P, (B) partially differentiated area in a teratocarcinoma from 477-l DD with cartilage, neural tubes, and an entodermal vesicle; (C) undifferentiated sarcomalike tumor from 978-l-6; (D) thymoma from BW5147. X100.

GM~~R,KNOWLES,AND SOLTER

Phenotype

of Teratocarcinoma

Hybrids

253

REFERENCES

strengthened by our findings of two different phenotypes in clonally derived hybrid cells from the same fusion (67, 72, and 978 series hybrids). The transition from the fibroblastoid to ECC morphology during passages in culture of single clones is a unique finding; growth of differentiated cells from phenotypically ECC hybrid clones is an expected consequence of differentiation and transformation. Though we can neither predict the phenotype of the product of a particular fusion, nor draw conclusions about the regulation of the phenotype in those hybrids, we do find that the phenotypic traits of a hybrid are expressed coordinately. That is, if the hybrid morphologically resembles ECC, it will express only ECC characteristics such as the ability to differentiate in vivo and in vitro, a specific composition of surface molecules and resistance to infection with certain viruses (Miller et al., 1977). Similarly, hybrids with differentiated phenotypes have characteristics that correlate with the appropriate morphology. It is possible then that there exists an integrated regulation of numerous genes which are coordinately expressed or suppressed, a concept derived from work of Fougere and Weiss (1978). The possibility of studying the pleiotropic effects of a single genetic mechanism is perhaps the most valuable contribution of this experimental system. In our efforts to approach the mechanism of this genetic control, we analyzed the chromosome composition of a series of hybrid subclones of ECC (477 series) and fibroblastoid (978 series) morphology; no obvious differences in the overall chromosome number of subclones from these two hybrid series were found. However comparison of chromosomes from the 67-2-5 subclones revealed that those of the fibroblastoid morphology contained a large number of chromosomal rearrangements. We could not account for the mechanism of appearance of fast-growing tumorigenic fibroblasts in the 67-2-5 ECC culture although it supports our notion that a single event can change the phenotype of an ECC. Similar results have been described in cloned teratocarcinoma stem cell lines, most notably the SIKR line (Evans, 1975; Martin and Evans, 1974; and our unpublished observations). Detailed karyotypic analysis of these 67-2-5 subclones and of subclones of the ECC and fibroblastoid morphology from the 978-l-5 H9 cell line should indicate if a specific chromosome can be implicated in the control of these major phenotypic transitions.

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We wish to thank Ru-Shya Liu, Adrienne Mihalek, and Daniela Simon for their excellent technical assistance. This research was supported by U.S. Public Health Research Grants CA-10815, CA-18470, CA-21069, and HD-1248’7; National Science Foundation Grant PCM79-17254and National Foundation-March of Dimes Grants l-301 and l-695. R. Gmlir was supported by a Fellowship from the Swiss National Science Foundation and B. B. Knowles by NIH Research Career Award AI-00053.

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