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Lack of expression of differentiation in mouse teratoma-fibroblast somatic cell hybrids
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Experimental Cell Research 76 (1973) 191-199
LACK OF EXPRESSION OF DIFFERENTIATION TERATOMA-FIBROBLAST J. JAMI, CHRISTIANE Institut de Recherches Scientifiques
SOMATIC FAILLY
IN MOUSE
CELL HYBRIDS
and EVELYNE RITZ
SW le Cancer du CNRS 94 Villejuif,
France
SUMMARY Somatic cell hybrids between multipotent mouse teratoma cells and mouse fibroblasts were established and isolated. The hybrid cells possess one chromosome set of each parental type and express major histocompatibility antigens of both strains of mice from which the parental cells were derived. Upon inoculation into Fl mice, hybrid cells produced tumors which were typical fibrosarcomas as were the tumors produced by the fibroblastic parental cell line. None of the well differentiated tissue types characteristic of the teratoma parent cell tumors were expressed in the hybrid tumors. The hybrid tumors possessed the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the cell populations inoculated. The possible role of ‘genie balance’ in phenotypic expression of cell hybrids is discussed.
arising teratocarcinomas, Transplantable spontaneously or induced, which contain multiple types of differentiated tissues have been described in the inbred mouse strain 129 by Stevens [l-3]. Single-cell transplantation experiments [4] as well as in vitro cultures of clonal populations derived from the ascitic form of the tumor have demonstrated that these tumors contain multipotential stem cells. The latter can be propagated as permanent lines of anaplastic cells or can differentiate into several well-defined types of tissue [5, 61. These stem cells (hereafter called teratoma cells) can be compared to cells of the early embryo which still maintain a:multiple developmental potential. We have undertaken the hybridization of such stem cells with cells which are already committed with respect to their tissue as a means to study some aspects of the processes involved in phenotype determination at the cellular level. 13 - 721815
Results of hybridization between teratoma cells and mouse fibroblasts are presented. Fibroblasts are usually characterized by collagen synthesis. We found that teratomafibroblast hybrids inoculated into mice grew into non-carcinomatous monomorphic tumors defined as fibrosarcomas. This result suggests that the hybrid cells express the potentiality shared by the two parental cells.
MATERIAL
AND METHODS
Cells The two cell types chosen for hybridization were: (a) fibroblast clone LM(TK-) Cl 1D derived from the L cell line of C3H inbred mice [7]. This clone (Cl 1D) is defective for thymidine-kinase and therefore resistant to bromodeoxyuridine. (b) Teratoma clone (SIKR) obtained from Dr M. Evans who derived it from the OTT 5568 tumor originally initiated by grafting a 3-day-old 129/Sv embryo into an adult 129-mouse testis [3]. The above tumor was shown to differentiate into a variety of tissue types when transplanted subcutaneously: neural tissue, epithelia, keratin pearls, pigmented cells, mesenchyme, cartilage, bone and striated muscle. Exptl Cell Res 76 (1973)
192 J. Jami et al.
‘Teratoma StKR
LM (T K-) 43 1D
Fig. 1. Karyograms of parental and hybrid cells. Arrows pointing to double constricted “D” chromosome.
The two cell lines were nronagated in culture in Dulbecco’s modified Eagle medium supplemented with 10 % calf serum and, for Cl 1D with 100 PM 5bromodeoxyuridine per ml. Hybrid colonies were selected in HAT (Eagle medium containing 100 uM hvuoxanthine. 10 uM amethopterin and-16 & thytmdine [S]. In HAT, Cl 1D cells are killed while SIKR cells and hybrids survive and can be isolated by cloning.
Hybridization The technique described by Davidson [9] was employed, using 1 x lo6 Cl 1D and 1 x lo3 SIKR cells, and 400 hemagglutinating units of UV-inactivated Sendai virus. Twentyfour hours after fusion, the cells were dispersed by trypsin and IO-fold dilutions were Exptl
Cell Res 76 (1973)
mated in 6 cm Falcon mastic dishes with 4 ml HAT. Medium was renewed every 4 days. Hybrid colonies were identified by their cell morphology which is distinct from SIKR colonies. Fifteen days after fusion, colonies were isolated in HAT, which was subsequently replaced by growth medium. Karyological preparations were made according to Rothfels & Siminovitch [lo] and stained by acetoorcein. The two parental cell populations SIKR and Cl 1D and their hybrids SIL 1 and SIL 6 were inoculated subcutaneously into groups of male mice of the following strains: C3H, 129/Sv and Fl of female 129/Sv x male C3H (129C3Fl). As soon as growing tumors reached l-2 cm, recipient mice were inoculated intraperitoneally with 0.75 mM colchicine. Animals were
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
193
Table 1. Karyotypes of parental cells and of their hybrids Number of chromosomes
Parental cells SIKR Cl 1D Hybrid cells Expected Observed SIL I SIL 6
Number of mitoses examined
Total
Telocentrics
Long metacentrics and submetacentrics
“D” marker
40 25
41 (38842) 52 (50-55)
41 (38-42) 44 (39947)
0 8 (7-l 1)
0 1
93 (88-97)
85 (77-89)
8 (7-11)
1
91 (83-93) 92 (90-93)
81 (7485) 85 (80-85)
9 (7-11) 7 (6-10)
1 1
25 25
sacrificed 12 h later and tumors were excised and divided into two parts. One part was fixed in Bouin fixative, embedded in paraffin and serially cut at 5 h&m.At every 100 sections, six consecutive section series were mounted and stained by hematoxylineosin. The preparations were examined for presence of differentiated tissues. The results were expressed as percentage frequency of a given tissue. The other part of the tumor was cut into fragments of 5 mm3 and treated twice with 3 % crude collagenase and 0.025 % trypsin in PBS for 45 min at 37°C. Karyological preparations were made with isolated cells.
RESULTS In vitro analysis of hybrids The frequency of colony forming hybrids was about 6 Y0 of the total SIKR cells plated (see Table 2. Inoculation
of parental
Methods). 135 presumed hybrid colonies were identified by their morphology. The two hybrid clones reported here SIL 1 and SIL 6 were isolated 15 days after fusion. Karyological examination (fig. 1) has shown for Cl 1D a modal number of 52 (50-55) chromosomes of which 44 (39-47) are telocentrics and 8 (7-l 1) long metacentrics and submetacentrics. Among the latter, one is characterized by a secondary constriction (“D” chromosome). SIKR contains a neardiploid modal number of 41 (38-42) chromosomes, all telocentrics. Cells in metaphase of the two hybrid clones have the sum of par-
and hybrid cells into mice
Tumor-bearing animalsb Cells inoculated’
Strain of host mice
SIKR
C3H 129C3Fl 129C3Fl C3H 1291s~ 129C3Fl C3H 1291s~ 129C3Fl C3H 1291s~ 129C3Fl
Cl ID SIL 1 SIL 6
Number
%
o/12 lo/lo IO/IO
0 100 100> 83 0 83 0 0 90
516 O/9 IO/l2 O/7 O/28 s/10 o/25 o/10 lo/lo
x 100
No. of tumors examined histologically
12 2
9 5
a In all cases 2.5 x 10” cells were inoculated subcutaneously. b As examined on the 28th day after inoculation. Exptl Cell Res 76 (1973)
194 J. Jami et al. ental chromosome complements; it is important to note that both quantitatively and qualitatively hybrid cells seem to possess only one chromosome set of each (table 1). These hybrids are therefore well-suited for the study of physiological interactions of the two parental genomes. In addition to the karyological data, the cell lines SIL 1 and SIL 6 exhibit transplantation properties typical of hybrid cells [l 11: they failed to grow in 129/Sv and C3H mice and gave tumors in 129C3Fl mice (table 3). Therefore, one can assume that these cells have 129 and C3H major histocompatibility antigens. Isoenzymatic patterns of carboxylic esterases, aldolases, lactate-dehydrogenases and glucose-6-phosphate dehydrogenases in parental cell extracts were studied by electrophoresis on acrylamide-agarose gels. The same isoenzymatic forms were present in Cl 1D and SIKR except for lactate-dehydrogenases, but their relative amounts were Table 3. Differentiation pendent SIKR tumors
index of four indeFrequency of tissues in tumor@
Tissue type
J20
3221 J222 J223
Carcinoma Mesenchyme Muscle Cartilage Bone Primitive ectoderm Neuroblastoma and neural tubules Keratinised stratified squamous enithelium Cuboidal epithelium Ciliated epithelium Glandular epithelium Parietal yolk sac Total number of sections examined
81 100 94 94 0 100
100 100 100 loo 82 54
100 100 0 13 0 36
100 100 18 64 18 18
100
loo
100
100
0 18 12 12 100
loo 36 45 21 100
36 18 18 0 36
100 100 73 9 18
32
22
22
22
a Percentage of sections with a given tissue out of the total number of sections examined (see Methods). Exptl
Cell Res 76 (1973)
different in the two parental cell extracts. They were also present in hybrid cell extracts in which the Cl 1D patterns were predominant, as estimated by the relative intensities of staining of isozymes. In addition to thosepresent in Cl lD, two faster molecular forms of lactate-dehydrogenases were found in SIKR; these two forms were not present in hybrid cell extracts.
Production of tumors and in vivo differentiation Upon inoculation into appropriate mice of 2.5 x lo6 SIKR, Cl 1D and hybrid cells, tumors were produced within 2-4 weeks (table 2). SIKR tumors contained several differentiated tissues (table 3). Though the method used to establish the differentiation index of teratoma tumors (see Methods) was a very useful way in demonstrating the multiplicity of differentiated tissues (figs 2-4), it does not account for the relative amounts of the tissues. For instance, carcinoma and neuroblastoma tissues were present in almost all sections of the tumors, but the index fails to show that carcinoma foci were very small as compared to the extensive areas of neuroblastoma. The ciliated epithelium indicated in table 3 came from one or two ducts for each tumor. Its frequency in serial sections may be related to differences in the angle of cutting. In contrast, Cl ID tumors are typical monomorphic fibrosarcomas (fig. 5). SIL 1 and SIL 6 hybrids produced tumors in almost all injected 129C3Fl mice (while no tumors were obtained from cells inoculated into C3H and 129 mice). All the hybrid tumors grew rapidly. Their lag period did not differ significantly from that of the tumors produced by the parental cells, arguing against the possibility that they were a product of selection of a small fraction of the initial cell population. Series of sections of 14 of these tumors
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
195
Ffgs 2-4. Sections from an SIKR tumor. The whole tumor consists of areas of various well-differentiated tissue types. Some well apparent tissues indicated by arrows: fig. 2a, keratin pearls; fig. 2b, 3b, cartilage nodules surrounded by mesenchyme,fig. 3c, bone; 3d, neuroblastic tissue with neural tubules. Hematoxylin-eosin, x 63. Fig. 4. An area of fig. 3 at higher magnification. x 160.
Exptl Cell Res 76 (1973)
196 J. Jami et al.
Fig. 5. Section from Cl 1D tumor. x 160. Fig. 6. Section from SIL 1 tumor. x 160. Both figures show a typical fibrosarcoma.
Exptl Cell Res 76 (1973)
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
197
these tumors have conserved the majority of the parental chromosomes. The presence of histocompatibility antigens of both C3H and 129 types supports this interpretation.
showed only fibrosarcoma (fig. 6). None of the other well-differentiated tissues observed in teratomas were seen. Noteworthy is the complete absence of carcinoma-like tissue. Karyology of tumor cells
DISCUSSION
Karyotypes of five out of the 14 tumors derived from hybrid cells inoculated into 129C3Fl mice, and which were examined histologically, were established. Results are presented in table 4. The modal numbers of chromosomes were 85 for SIL 1 tumors and 67 for the XL 6 tumor, as compared to 91 and 92 respectively established for the inoculated cells. Thus, in all cases examined, the number of chromosomes in tumor cells was reduced in comparison with the hybrid cultures before inoculation. The number of long metacentric and submetacentric chromosomes corresponds to that of Cl ID 1s. Although one cannot distinguish morphologically between the telocentric chromosomes of the two parents, their approximate sum is found in the hybrid metaphases. Thus we found no evidence of major chromosomal rearrangement implicating elimination of a great number of chromosomes of one parental cell and polyploidy of the other parental genome. Rather, the data indicate that the cells of
Cell hybrids (SIL) of mouse teratoma (SIKR) and mouse fibroblast (Cl 1D) inoculated into 129C3Fl mice produce tumors which are typical fibrosarcomas. The cells of these hybrid tumors possess the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the populations inoculated. These results raise a few questions. (1) May one consider the fibrosarcoma as the phenotypic expression of teratoma-fibroblast hybrid population possessing the entire complement of parental chromosomes? Parental Cl 1D tumors and those produced by hybrids have the same phenotype. Thus, since the hybrids did not express the tissuephenotype observed in SIKR tumors, we have focused our attention on the question of presence of the SIKR chromosomes. Complete SIKR genome is most likely not present in each of the cells of the hybrid-derived tu-
Table 4. Karyotypes of tumors obtained from hybrid cells
SILa SIL 1 tumorsb 548 J52 J56 J57 SIL 6a SIL 6 tumorsb J50
Number of chromosomes
No. of mitoses examined
Total
25
91 (83-93)
81 (74-85)
9 (7-11)
:i 25 23 25
84 (79-88) 86 (75-89) 85 (81-91) 86 (82-92) 92 (90-93)
75 (71-78) 76 (66-80) 74 (74-83) 76 (72-81) 85 (80-85)
9 (8-10) 9 (7-12) 9 (7-11) 10 (8-12) 7 (6-10)
0 (O-1) 1 (O-l) 1 (O-1) 1 (O-l) 1
32
67 (61-92)
63 (54-68)
6 (2-12)
1 (O-2)
Telocentrics
Long metacentrics and submetacentrics
“D” marker 1
a In vitro maintained hybrid cell culture. Chromosome analysis made shortly prior to inoculation into mice. ’ Chromosome analysis of cells from dissociated tumors (see Methods). Exptl Cell Res 76 (197 3
198 J. Jami et al. mors. However, four out of five tumors investigated have shown chromosome scores close to the initial population, having lost only 5-7 telocentrics. Since chromosome loss from hybrid cells is a random event [12], and since it is impossible to distinguish SIKR chromosomes from Cl 1D telocentrics, the observed numbers can be due to chromosome losses of either one or both parents. Along the same ime of reasoning, one could expect to find on the level of the entire tumor cell populations at least some cells containing all the SIKR chromosomes. Examination of serial sections from 14 independent tumors did not permit identification of any other tissue-type than fibrosarcoma. Finch & Ephrussi reported similar observations [ 131. Thus, if indeed cells with complete SIKR genome were among the hybrids with Cl lD, then either fibrosarcoma is their phenotypic expression, or they were present in insufficient numbers to be noticed. Our results therefore indicate that hybridization of a multipotential teratoma cell with a fibroblast results in the extinction of expression of any phenotype other than the one common to both parental cells. (2) Is collagen synthesis a differentiated function? Collagen synthesis was demonstrated in many different cell types. However, fibroblasts were shown to synthezise significantly more collagen than any other cell examined [14, 151. Thus, it is not the synthesis of collagen but its abundance with its characteristic histological picture that is accepted as the feature of mesenchyme. Large deposits of collagen were seen in Cl 1D tumors as well as in Cl lD-SIKR-hybrid tumors. (3) What is the significance of the exclusive collagen synthesis as a phenotypic expression of the SIL hybrid-cell population? Cell hybridization has been used in an attempt to elucidate the mechanisms underExpel Cell Res 76 (1973)
lying the phenomenon of restricted phenotypic expression of differentiated cells. A persistent synthesis of homologous proteins was shown in the case of hybrids between cells expressing same function before fusion [16-181. In the case of fusion between cells varying qualitatively with respect to their expression of a differentiated function, two categories of hybrids were observed:
(a) those in which the expression specific to one of the parental cells was suppressed [19-241. (b) those in which the expression of a specific parental function was maintained [25-271. It is important to note that in all so far documented hybrids the parental cell expressing the investigated function was at least hypotetraploid. Moreover, in the latter category, the functional hybrids generally contained more than one set of chromosomes derived from the functional parent-cell versus a single copy of the other. A particular case of interest were the hybrids obtained with rat hepatoma and mouse fibroblast in which synthesis of mouse albumin was induced [25]. Thus, the notion of ‘genie balance’ implying the importance of a quantitative relationship between genes involved in the expression of a given function was brought forward [27]. Coming back to our results, one should point out that while SIKR is near-diploid, Cl 1D is hypotetraploid and thus the genie rapport in the hybrids is in favor of the fibroblast. The phenotypic expression of SIL hybrids allows including them in the first category of hybrids discussed above. The application of the recently introduced techniques of individual chromosome identification [28-301 should allow study of the correlation between the phenotypic expression of hybrids and the relative numbers of homologous chromosomes. Further study of hybrids derived from diploid parental cells
Lack of differentiation
with different chromosomal ought to be performed.
composition
We thank Dr Y. Berwald-Netter for stimulating discussions and help in the preparation of the manuscript, and Mrs M. Vicomte for excellent assistance with the histological preparations. J. J. is attache de Recherche at I’INSERM.
REFERENCES Stevens, L C, J natl cancer inst 20 (1958) 1257. - J embrvol exutl mornhol 20 (1968) 329. - Dev bcol 21 (1970) 364. _ ’ Kleinsmith, L J & Pierce, G B, Cancer res 24 (1964) 1544. 5. Rosenthal, M D, Wishnow, R M & Sato, G H, J natl cancer inst 44 (1970) 1001. 6. Kahan, B W & Ephrussi, B, J natl cancer inst 44 (1970) 1015. 7. Kit; S, Dubbs, D R, Piekarski, L J & Hsu, T C, Exptl cell res 31 (1963) 297. Littlefield, J W, Science 145 (1964) 709. ;. Davidson, R L, Exptl cell res 55 (1969) 424. 10: Rothfels, K & Siminovitch, L, Stain technol 33 (1958) 73. 11. Spencer, R A, Hauschka, T S, Amos, D B & Ephrussi, B, J natl cancer inst 33 (1964) 893. 12. Ephrussi, B, Scaletta, L J, Stenchever, M A & Yoshida, M, Symp int sot cell biol 3 (1964) 13. 13. Finch, B W & Ephrussi, B, Proc natl acad sci US 57 (1967) 615. 14. Green, H, Goldberg, B & Todaro, G J, Nature 212 (1966) 631. 1. 2. 3. 4.
in mouse teratoma-fibroblast
hybrids
199
15. Green, H & Goldberg, B, Symp int sot cell biol 7 (1968) 123. 16. Green, H, Ephrussi, B, Yoshida, M & Hamerman, D, Proc natl acad sci US 55 (1966) 41. 17. I$lefield, J W, Proc natl acad sci US 62 (1969) 18. Simoni, G, Balacco, S, Nuzzo, F, Larizza, L & De Carli, L, Atti ass genet ital 15 (1970) 130. 19. Davidson, R L, Ephrussi, B & Yamamoto, K, Proc natl acad sci US 56 (1966) 1437. 20. Silagi, S, Cancer res 27 (1967) 1953. 21. Sonnenschein, C, Richardson, U I & Tashjian A H Jr, Exptl cell res 69 (1971) 336. 22. Schneider, J A & Weiss, M C, Proc natl acad sci US 68 (1971) 127. 23. Mohit, B & Fan, K, Science 171 (1971) 75. 24. Coffino, P, Knowles, B, Nathenson, S G & Scharf, M D, Nature new biol 231 (1971) 87. 25. Peterson. J A & Weiss. M C. Proc natl acad sci US 69 (1972) 571. 26. Minna, J, Glazer, D & Nirenberg, M, Nature new biol 235 (1972) 225. 27. Fougere, C, Ruiz, F & Ephrussi, B, Proc natl acad sci US 69 (1972) 330. 28. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 29. Sumner, \ A T, Evans, H J & Buckland, R A, Nature new biol 232 (1971) 31. 30. Dutrillaux, B, De Grouchy, J, Finas, C & Lejeune, J, Compt rend acad sci 273 (1971) 587.
Received May 5, 1972 Revised version received July 3, 1972
Exptl Cell Res 76 (1973
Experimental Cell Research 76 (1973) 191-199
LACK OF EXPRESSION OF DIFFERENTIATION TERATOMA-FIBROBLAST J. JAMI, CHRISTIANE Institut de Recherches Scientifiques
SOMATIC FAILLY
IN MOUSE
CELL HYBRIDS
and EVELYNE RITZ
SW le Cancer du CNRS 94 Villejuif,
France
SUMMARY Somatic cell hybrids between multipotent mouse teratoma cells and mouse fibroblasts were established and isolated. The hybrid cells possess one chromosome set of each parental type and express major histocompatibility antigens of both strains of mice from which the parental cells were derived. Upon inoculation into Fl mice, hybrid cells produced tumors which were typical fibrosarcomas as were the tumors produced by the fibroblastic parental cell line. None of the well differentiated tissue types characteristic of the teratoma parent cell tumors were expressed in the hybrid tumors. The hybrid tumors possessed the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the cell populations inoculated. The possible role of ‘genie balance’ in phenotypic expression of cell hybrids is discussed.
arising teratocarcinomas, Transplantable spontaneously or induced, which contain multiple types of differentiated tissues have been described in the inbred mouse strain 129 by Stevens [l-3]. Single-cell transplantation experiments [4] as well as in vitro cultures of clonal populations derived from the ascitic form of the tumor have demonstrated that these tumors contain multipotential stem cells. The latter can be propagated as permanent lines of anaplastic cells or can differentiate into several well-defined types of tissue [5, 61. These stem cells (hereafter called teratoma cells) can be compared to cells of the early embryo which still maintain a:multiple developmental potential. We have undertaken the hybridization of such stem cells with cells which are already committed with respect to their tissue as a means to study some aspects of the processes involved in phenotype determination at the cellular level. 13 - 721815
Results of hybridization between teratoma cells and mouse fibroblasts are presented. Fibroblasts are usually characterized by collagen synthesis. We found that teratomafibroblast hybrids inoculated into mice grew into non-carcinomatous monomorphic tumors defined as fibrosarcomas. This result suggests that the hybrid cells express the potentiality shared by the two parental cells.
MATERIAL
AND METHODS
Cells The two cell types chosen for hybridization were: (a) fibroblast clone LM(TK-) Cl 1D derived from the L cell line of C3H inbred mice [7]. This clone (Cl 1D) is defective for thymidine-kinase and therefore resistant to bromodeoxyuridine. (b) Teratoma clone (SIKR) obtained from Dr M. Evans who derived it from the OTT 5568 tumor originally initiated by grafting a 3-day-old 129/Sv embryo into an adult 129-mouse testis [3]. The above tumor was shown to differentiate into a variety of tissue types when transplanted subcutaneously: neural tissue, epithelia, keratin pearls, pigmented cells, mesenchyme, cartilage, bone and striated muscle. Exptl Cell Res 76 (1973)
192 J. Jami et al.
‘Teratoma StKR
LM (T K-) 43 1D
Fig. 1. Karyograms of parental and hybrid cells. Arrows pointing to double constricted “D” chromosome.
The two cell lines were nronagated in culture in Dulbecco’s modified Eagle medium supplemented with 10 % calf serum and, for Cl 1D with 100 PM 5bromodeoxyuridine per ml. Hybrid colonies were selected in HAT (Eagle medium containing 100 uM hvuoxanthine. 10 uM amethopterin and-16 & thytmdine [S]. In HAT, Cl 1D cells are killed while SIKR cells and hybrids survive and can be isolated by cloning.
Hybridization The technique described by Davidson [9] was employed, using 1 x lo6 Cl 1D and 1 x lo3 SIKR cells, and 400 hemagglutinating units of UV-inactivated Sendai virus. Twentyfour hours after fusion, the cells were dispersed by trypsin and IO-fold dilutions were Exptl
Cell Res 76 (1973)
mated in 6 cm Falcon mastic dishes with 4 ml HAT. Medium was renewed every 4 days. Hybrid colonies were identified by their cell morphology which is distinct from SIKR colonies. Fifteen days after fusion, colonies were isolated in HAT, which was subsequently replaced by growth medium. Karyological preparations were made according to Rothfels & Siminovitch [lo] and stained by acetoorcein. The two parental cell populations SIKR and Cl 1D and their hybrids SIL 1 and SIL 6 were inoculated subcutaneously into groups of male mice of the following strains: C3H, 129/Sv and Fl of female 129/Sv x male C3H (129C3Fl). As soon as growing tumors reached l-2 cm, recipient mice were inoculated intraperitoneally with 0.75 mM colchicine. Animals were
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
193
Table 1. Karyotypes of parental cells and of their hybrids Number of chromosomes
Parental cells SIKR Cl 1D Hybrid cells Expected Observed SIL I SIL 6
Number of mitoses examined
Total
Telocentrics
Long metacentrics and submetacentrics
“D” marker
40 25
41 (38842) 52 (50-55)
41 (38-42) 44 (39947)
0 8 (7-l 1)
0 1
93 (88-97)
85 (77-89)
8 (7-11)
1
91 (83-93) 92 (90-93)
81 (7485) 85 (80-85)
9 (7-11) 7 (6-10)
1 1
25 25
sacrificed 12 h later and tumors were excised and divided into two parts. One part was fixed in Bouin fixative, embedded in paraffin and serially cut at 5 h&m.At every 100 sections, six consecutive section series were mounted and stained by hematoxylineosin. The preparations were examined for presence of differentiated tissues. The results were expressed as percentage frequency of a given tissue. The other part of the tumor was cut into fragments of 5 mm3 and treated twice with 3 % crude collagenase and 0.025 % trypsin in PBS for 45 min at 37°C. Karyological preparations were made with isolated cells.
RESULTS In vitro analysis of hybrids The frequency of colony forming hybrids was about 6 Y0 of the total SIKR cells plated (see Table 2. Inoculation
of parental
Methods). 135 presumed hybrid colonies were identified by their morphology. The two hybrid clones reported here SIL 1 and SIL 6 were isolated 15 days after fusion. Karyological examination (fig. 1) has shown for Cl 1D a modal number of 52 (50-55) chromosomes of which 44 (39-47) are telocentrics and 8 (7-l 1) long metacentrics and submetacentrics. Among the latter, one is characterized by a secondary constriction (“D” chromosome). SIKR contains a neardiploid modal number of 41 (38-42) chromosomes, all telocentrics. Cells in metaphase of the two hybrid clones have the sum of par-
and hybrid cells into mice
Tumor-bearing animalsb Cells inoculated’
Strain of host mice
SIKR
C3H 129C3Fl 129C3Fl C3H 1291s~ 129C3Fl C3H 1291s~ 129C3Fl C3H 1291s~ 129C3Fl
Cl ID SIL 1 SIL 6
Number
%
o/12 lo/lo IO/IO
0 100 100> 83 0 83 0 0 90
516 O/9 IO/l2 O/7 O/28 s/10 o/25 o/10 lo/lo
x 100
No. of tumors examined histologically
12 2
9 5
a In all cases 2.5 x 10” cells were inoculated subcutaneously. b As examined on the 28th day after inoculation. Exptl Cell Res 76 (1973)
194 J. Jami et al. ental chromosome complements; it is important to note that both quantitatively and qualitatively hybrid cells seem to possess only one chromosome set of each (table 1). These hybrids are therefore well-suited for the study of physiological interactions of the two parental genomes. In addition to the karyological data, the cell lines SIL 1 and SIL 6 exhibit transplantation properties typical of hybrid cells [l 11: they failed to grow in 129/Sv and C3H mice and gave tumors in 129C3Fl mice (table 3). Therefore, one can assume that these cells have 129 and C3H major histocompatibility antigens. Isoenzymatic patterns of carboxylic esterases, aldolases, lactate-dehydrogenases and glucose-6-phosphate dehydrogenases in parental cell extracts were studied by electrophoresis on acrylamide-agarose gels. The same isoenzymatic forms were present in Cl 1D and SIKR except for lactate-dehydrogenases, but their relative amounts were Table 3. Differentiation pendent SIKR tumors
index of four indeFrequency of tissues in tumor@
Tissue type
J20
3221 J222 J223
Carcinoma Mesenchyme Muscle Cartilage Bone Primitive ectoderm Neuroblastoma and neural tubules Keratinised stratified squamous enithelium Cuboidal epithelium Ciliated epithelium Glandular epithelium Parietal yolk sac Total number of sections examined
81 100 94 94 0 100
100 100 100 loo 82 54
100 100 0 13 0 36
100 100 18 64 18 18
100
loo
100
100
0 18 12 12 100
loo 36 45 21 100
36 18 18 0 36
100 100 73 9 18
32
22
22
22
a Percentage of sections with a given tissue out of the total number of sections examined (see Methods). Exptl
Cell Res 76 (1973)
different in the two parental cell extracts. They were also present in hybrid cell extracts in which the Cl 1D patterns were predominant, as estimated by the relative intensities of staining of isozymes. In addition to thosepresent in Cl lD, two faster molecular forms of lactate-dehydrogenases were found in SIKR; these two forms were not present in hybrid cell extracts.
Production of tumors and in vivo differentiation Upon inoculation into appropriate mice of 2.5 x lo6 SIKR, Cl 1D and hybrid cells, tumors were produced within 2-4 weeks (table 2). SIKR tumors contained several differentiated tissues (table 3). Though the method used to establish the differentiation index of teratoma tumors (see Methods) was a very useful way in demonstrating the multiplicity of differentiated tissues (figs 2-4), it does not account for the relative amounts of the tissues. For instance, carcinoma and neuroblastoma tissues were present in almost all sections of the tumors, but the index fails to show that carcinoma foci were very small as compared to the extensive areas of neuroblastoma. The ciliated epithelium indicated in table 3 came from one or two ducts for each tumor. Its frequency in serial sections may be related to differences in the angle of cutting. In contrast, Cl ID tumors are typical monomorphic fibrosarcomas (fig. 5). SIL 1 and SIL 6 hybrids produced tumors in almost all injected 129C3Fl mice (while no tumors were obtained from cells inoculated into C3H and 129 mice). All the hybrid tumors grew rapidly. Their lag period did not differ significantly from that of the tumors produced by the parental cells, arguing against the possibility that they were a product of selection of a small fraction of the initial cell population. Series of sections of 14 of these tumors
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
195
Ffgs 2-4. Sections from an SIKR tumor. The whole tumor consists of areas of various well-differentiated tissue types. Some well apparent tissues indicated by arrows: fig. 2a, keratin pearls; fig. 2b, 3b, cartilage nodules surrounded by mesenchyme,fig. 3c, bone; 3d, neuroblastic tissue with neural tubules. Hematoxylin-eosin, x 63. Fig. 4. An area of fig. 3 at higher magnification. x 160.
Exptl Cell Res 76 (1973)
196 J. Jami et al.
Fig. 5. Section from Cl 1D tumor. x 160. Fig. 6. Section from SIL 1 tumor. x 160. Both figures show a typical fibrosarcoma.
Exptl Cell Res 76 (1973)
Lack of differentiation
in mouse teratoma-fibroblast
hybrids
197
these tumors have conserved the majority of the parental chromosomes. The presence of histocompatibility antigens of both C3H and 129 types supports this interpretation.
showed only fibrosarcoma (fig. 6). None of the other well-differentiated tissues observed in teratomas were seen. Noteworthy is the complete absence of carcinoma-like tissue. Karyology of tumor cells
DISCUSSION
Karyotypes of five out of the 14 tumors derived from hybrid cells inoculated into 129C3Fl mice, and which were examined histologically, were established. Results are presented in table 4. The modal numbers of chromosomes were 85 for SIL 1 tumors and 67 for the XL 6 tumor, as compared to 91 and 92 respectively established for the inoculated cells. Thus, in all cases examined, the number of chromosomes in tumor cells was reduced in comparison with the hybrid cultures before inoculation. The number of long metacentric and submetacentric chromosomes corresponds to that of Cl ID 1s. Although one cannot distinguish morphologically between the telocentric chromosomes of the two parents, their approximate sum is found in the hybrid metaphases. Thus we found no evidence of major chromosomal rearrangement implicating elimination of a great number of chromosomes of one parental cell and polyploidy of the other parental genome. Rather, the data indicate that the cells of
Cell hybrids (SIL) of mouse teratoma (SIKR) and mouse fibroblast (Cl 1D) inoculated into 129C3Fl mice produce tumors which are typical fibrosarcomas. The cells of these hybrid tumors possess the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the populations inoculated. These results raise a few questions. (1) May one consider the fibrosarcoma as the phenotypic expression of teratoma-fibroblast hybrid population possessing the entire complement of parental chromosomes? Parental Cl 1D tumors and those produced by hybrids have the same phenotype. Thus, since the hybrids did not express the tissuephenotype observed in SIKR tumors, we have focused our attention on the question of presence of the SIKR chromosomes. Complete SIKR genome is most likely not present in each of the cells of the hybrid-derived tu-
Table 4. Karyotypes of tumors obtained from hybrid cells
SILa SIL 1 tumorsb 548 J52 J56 J57 SIL 6a SIL 6 tumorsb J50
Number of chromosomes
No. of mitoses examined
Total
25
91 (83-93)
81 (74-85)
9 (7-11)
:i 25 23 25
84 (79-88) 86 (75-89) 85 (81-91) 86 (82-92) 92 (90-93)
75 (71-78) 76 (66-80) 74 (74-83) 76 (72-81) 85 (80-85)
9 (8-10) 9 (7-12) 9 (7-11) 10 (8-12) 7 (6-10)
0 (O-1) 1 (O-l) 1 (O-1) 1 (O-l) 1
32
67 (61-92)
63 (54-68)
6 (2-12)
1 (O-2)
Telocentrics
Long metacentrics and submetacentrics
“D” marker 1
a In vitro maintained hybrid cell culture. Chromosome analysis made shortly prior to inoculation into mice. ’ Chromosome analysis of cells from dissociated tumors (see Methods). Exptl Cell Res 76 (197 3
198 J. Jami et al. mors. However, four out of five tumors investigated have shown chromosome scores close to the initial population, having lost only 5-7 telocentrics. Since chromosome loss from hybrid cells is a random event [12], and since it is impossible to distinguish SIKR chromosomes from Cl 1D telocentrics, the observed numbers can be due to chromosome losses of either one or both parents. Along the same ime of reasoning, one could expect to find on the level of the entire tumor cell populations at least some cells containing all the SIKR chromosomes. Examination of serial sections from 14 independent tumors did not permit identification of any other tissue-type than fibrosarcoma. Finch & Ephrussi reported similar observations [ 131. Thus, if indeed cells with complete SIKR genome were among the hybrids with Cl lD, then either fibrosarcoma is their phenotypic expression, or they were present in insufficient numbers to be noticed. Our results therefore indicate that hybridization of a multipotential teratoma cell with a fibroblast results in the extinction of expression of any phenotype other than the one common to both parental cells. (2) Is collagen synthesis a differentiated function? Collagen synthesis was demonstrated in many different cell types. However, fibroblasts were shown to synthezise significantly more collagen than any other cell examined [14, 151. Thus, it is not the synthesis of collagen but its abundance with its characteristic histological picture that is accepted as the feature of mesenchyme. Large deposits of collagen were seen in Cl 1D tumors as well as in Cl lD-SIKR-hybrid tumors. (3) What is the significance of the exclusive collagen synthesis as a phenotypic expression of the SIL hybrid-cell population? Cell hybridization has been used in an attempt to elucidate the mechanisms underExpel Cell Res 76 (1973)
lying the phenomenon of restricted phenotypic expression of differentiated cells. A persistent synthesis of homologous proteins was shown in the case of hybrids between cells expressing same function before fusion [16-181. In the case of fusion between cells varying qualitatively with respect to their expression of a differentiated function, two categories of hybrids were observed:
(a) those in which the expression specific to one of the parental cells was suppressed [19-241. (b) those in which the expression of a specific parental function was maintained [25-271. It is important to note that in all so far documented hybrids the parental cell expressing the investigated function was at least hypotetraploid. Moreover, in the latter category, the functional hybrids generally contained more than one set of chromosomes derived from the functional parent-cell versus a single copy of the other. A particular case of interest were the hybrids obtained with rat hepatoma and mouse fibroblast in which synthesis of mouse albumin was induced [25]. Thus, the notion of ‘genie balance’ implying the importance of a quantitative relationship between genes involved in the expression of a given function was brought forward [27]. Coming back to our results, one should point out that while SIKR is near-diploid, Cl 1D is hypotetraploid and thus the genie rapport in the hybrids is in favor of the fibroblast. The phenotypic expression of SIL hybrids allows including them in the first category of hybrids discussed above. The application of the recently introduced techniques of individual chromosome identification [28-301 should allow study of the correlation between the phenotypic expression of hybrids and the relative numbers of homologous chromosomes. Further study of hybrids derived from diploid parental cells
Lack of differentiation
with different chromosomal ought to be performed.
composition
We thank Dr Y. Berwald-Netter for stimulating discussions and help in the preparation of the manuscript, and Mrs M. Vicomte for excellent assistance with the histological preparations. J. J. is attache de Recherche at I’INSERM.
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Received May 5, 1972 Revised version received July 3, 1972
Exptl Cell Res 76 (1973