THE ANALYSIS OF MALIGNANCY BY CELL FUSION II. HYBRIDS BETWEEN EHRLICH CELLS AND NORMAL DIPLOID CELLS

Transcription

1 J. Cell Sci. 8, (97) 673 Printed in Great Britain THE ANALYSIS OF MALIGNANCY BY CELL FUSION II. HYBRIDS BETWEEN EHRLICH CELLS AND NORMAL DIPLOID CELLS U. BREGULA,* G. KLEIN Department of Tumor Biology, Karolinska Institutet, 4 Stockholm 6, Sweden AND H.HARRIS Sir William Dunn School of Pathology, University of Oxford, Oxford OXi 3 RE, England SUMMARY When Ehrlich ascites cells were fused with diploid fibroblasts, isolated directly from the animal, the resulting hybrid cells regularly produced progressive tumours. However, an analysis of a range of clonal populations of these hybrid cells, each derived from a separate primary fusion, revealed that the chromosomal constitution of these cells was highly unstable; all cell populations were found to have already undergone substantial chromosome losses by the time enough cells were available to permit chromosomal analysis. Thus, although these hybrid cells were highly tumorigenic, the tumours arising from them were not composed of cells with complete parental chromosome sets, but of cells from which some chromosomes had been eliminated. INTRODUCTION In the present paper the characteristics of hybrids between Ehrlich ascites cells and normal diploid fibroblasts are described. Three wild type populations, each derived from different fusion experiments, and a number of clonal populations, each derived from a separate primary fusion, were examined. MATERIALS AND METHODS Cell types The fibroblasts were obtained by trypsinization of -day-old embryos from CBA mice bearing the T6T6 chromosomal translocation (Ford, Hamerton, Barnes & Loutit, 956). The primary cultures were grown for a few days in plastic flasks and then transferred by trypsinization to fresh flasks. The secondary cultures, and occasionally later subcultures, were used for the fusion experiments. Fellow of the International Agency for Research on Cancer, on leave from the Institute of Oncology, Warsaw, Poland.

2 674 U- Bregula, G. Klein and H. Harris Cell fusion A mixed suspension of 5 x ' Ehrlich cells, taken directly from the mouse peritoneal cavity, and 5 x 6 fibroblasts, harvested from monolayers by trypsinization, were treated with haemagglutinating units of inactivated Sendai virus. The fused cells were introduced into plastic flasks and grown on the floor of the flasks in Dulbecco's medium. Selection of hybrids The Ehrlich cells, which did not adhere to thefloorof the culture flasks, were eliminated by frequent changes of medium during the first few days. The Ehrlich/fibroblast hybrid cells adhered to the plastic surface much less tenaciously than the fibroblasts themselves and frequently came away from the floor of the flask at mitosis. When the cultures had become confluent, theflaskswere shaken vigorously and the cells in suspension transferred to a fresh flask. Subcultivation by this procedure led to progressive enrichment of the cultures with hybrid cells and eventually to the production of pure cultures of hybrid cells. In order to obtain clonal populations of hybrid cells, each derived from a separate primary fusion, the fused cells were trypsinized after 3 days' cultivation and the trypsinized cells seeded into 2-in. (5-cm) diameter plastic Petri dishes at a concentration of 5 cells per dish. The dishes were then incubated until visible clones appeared, usually a period of about 2 weeks. The clones were then examined microscopically and those composed of cells with a morphology clearly different from that of the fibroblasts themselves were selected for harvesting. Each selected clone was trypsinized into a small metal cylinder attached to thefloorof the dish around the clone by a film of silicone grease, and the resulting cell suspension was introduced into a small plastic flask. Each clone was then grown up to give a population of cells large enough for karyotypic analysis and assay in vivo. The clonal populations were first assayed for growth in vivo just as soon as enough cells were available to provide an adequate inoculum; but subsequent assays were carried out on populations maintained by repeated subcultivation in vitro. Assay for tumorigenicity Most assays were carried out in newborn CBA mice given 4 rd (4 J kg" ) of X-irradiation. A few tests were also made in unirradiated newborn CBA mice. Inocula of io 4 to 2 x io cells were injected subcutaneously and the animals were scanned in the usual way for the appearance of tumours. RESULTS Growth of Ehrlichlfibroblast hybrids in vivo All hybrid populations were identified by the presence of both the Ehrlich and the T6 chromosomal markers in at least some of the cells in each population. The results of the assays for growth in vivo are given in Table. It will be seen that all wild type and clonal populations were highly tumorigenic. Although there was some variation in take incidence from one clonal population to another, none of the clones showed a take incidence lower than 6%. There were, moreover, some differences in the growth rates of the tumours produced by different cell populations. Chromosomal constitution of tlie hybrid cells The high take incidence of all the Ehrlich/fibroblast hybrids would, at first glance, lead one to conclude that in this combination the malignancy of the Ehrlich cell was dominant. However, an examination of the chromosomal constitution of the hybrid cells revealed a situation of greater complexity. Tables 2 and 3 and Fig. show the

3 Analysis of malignancy. II 675 chromosomal constitutions of the 3 wild type populations and of all the clonal populations. All of these analyses were done just as soon as populations large enough for the purpose could be obtained. The chromosome number to be expected in a hybrid cell resulting from the fusion of one modal Ehrlich cell and one fibroblast would be about 6; but it will be seen that none of the Ehrlich/fibroblast hybrids showed this modal chromosome number. Apart from populations composed of clearly polyploid clones wild type Clone. Clone 2 g iio l o ^3 52 ^ i I 3 I 2 js S "o S,2 g u ,, Clone 3 I I L. Clone 5 i I I. I I I I I L II Clone 7B Clone 8 done 9 Clone Clone K>" >245 Chromosome number Fig.. Distribution of chromosomes in clonal populations of Ehrlich/fibroblast hybrids. The expected chromosome number for a hybrid cell resulting from the fusion of one modal Ehrlich cell and one diploid fibroblast would be 6. Except for grossly polyploid clones, all the hybrid populations have modal chromosome numbers well below that to be expected from the sum of the 2 parental chromosome sets.

4 676 U. Bregula, G. Klein and H. Harris (clones 4, 9 and ), all these hybrids, as soon as they could be analysed, already had between 5 and fewer chromosomes than would be expected in a hybrid containing complete sets of chromosomes from the 2 parent cells; and it has not, in fact, proved possible to obtain a hybrid cell population with an unreduced chromosome complement from this particular combination of parents. This result is very different from that obtained when the Ehrlich, and other tumour, cells were fused with the L cell derivatives, when hybrid cells having a chromosome number approximating to the sum of the 2 parental modes were easily obtained. It thus appears that the chromosomal constitution of the Ehrlich/fibroblast hybrids is much less stable than that of the hybrids between the Ehrlich cells and the L cell derivatives; and it is clear that a large number of chromosomes are eliminated from the Ehrlich/fibroblast hybrids in the early cell divisions. This observation is of some interest for it demonstrates that rapidity of chromosome elimination in hybrid cells can be determined by factors other than differences in the species of origin of the parent cells. Even intraspecific hybrids may undergo drastic reduction in chromosome number when certain combinations of parent cells are used. The failure to produce any Ehrlich/fibroblast hybrids containing the expected chromosome number for the sum of the 2 parental chromosome sets makes it impossible to draw any conclusion about the dominance or recessiveness of the malignant trait in this combination. Since these hybrids eliminate chromosomes very readily, the chances of selecting malignant segregants from the hybrid cell population will clearly be much greater than in hybrids where the complete chromosome sets of the 2 parent cells tend to be maintained for long periods. This could well explain the high take incidences obtained with Ehrlich/fibroblast hybrids. But the question arises why loss of chromosomes in vitro should result in a high incidence of malignant cells in Ehrlich/ fibroblast cultures, when loss of chromosomes in vitro does not increase the incidence of malignant cells in Ehrlich/Ao, cultures. The answer no doubt lies in the nature of the chromosomes lost in the two cases. We have seen that the A9 chromosomes (at least the bi-armed ones) tend to be retained in Ehrlich/A9 hybrids on cultivation in vitro. But in the case of hybrids between Ehrlich cells and freshly isolated mouse fibroblasts, which are, unlike A 9 cells, poorly adapted to growth in vitro, it is quite possible that the fibroblast chromosomes are preferentially eliminated, or, at least, are not preferentially retained. The absence of marker chromosomes other than the T6 translocation in the fibroblasts frustrates direct investigation of this point; but there is now substantial evidence that when cells poorly adapted to growth in vitro are fused with others, replication of DNA may lag in the nuclei of the poorly adapted cells and their chromosomes may be preferentially eliminated (Johnson & Harris, 969; Harris, 97; Johnson & Rao, 97; Johnson, Rao & Hughes, 97; Schwartz, Cook & Harris, 97). That some fibroblast chromosomes are lost from the Ehrlich/fibroblast hybrids is shown by the absence of at least one of the T 6 chromosomes in the great majority of the hybrid cells examined. More decisive observations on the role of chromosome loss in determining the incidence of malignant cells in hybrid cell cultures will be presented in the following paper.

5 Tumours produced from Ehrlich/fibroblast hybrids Analysis of malignancy. II 677 Analysis of the chromosomes of the tumours produced by the injection of these hybrids also revealed the operation of in vivo selection, although the results were not as dramatic as those seen with the Ehrlich/Ao. hybrids. The results of some typical analyses are shown in Figs In some cases, the modal chromosome number of the 3 3 u 2? IE O S "5 2 V -C DO U s c in vitro (26--69) J I tumour (3-2-69) iso Chromosome number Fig. 2. Distribution of chromosomes in a clonal population of Ehrlich/fibroblast hybrids growing in vitro and in a tumour arising from these hybrids. In this case there is only a slight reduction in the modal chromosome number of the cells in the tumour; but the chromosome number of the hybrid cells in vitro was already substantially lower than that to be expected from the sum of the 2 parental chromosome sets. 2 " in vitro (6-2-69) i i il i i M..U IB i 2 _ tumour (-2-7) (CBA mouse) i i l, Mill II 2 _ tumour (2-3-7) (C3HxCBA mouse) l i i I.... II, 2 3 U >2 Chromosome number Fig. 3. Distribution of chromosomes in a clonal population of Ehrlich/fibroblast hybrids growing in vitro and in tumours arising from these hybrids. The hybrid cells in this case were highly polyploid. The tumours derived from them show a reduction in chromosome number relative to that of the cells injected.. tumour cells was little different from that of the hybrid population injected into the animal (Fig. 2). This finding is not, however, of any special significance, since the hybrid cells had already undergone drastic chromosomal losses in vitro. In other cases, the cells in the tumour showed a reduction in modal chromosome number relative to

6 678 U. Bregula, G. Klein and H. Harris that of the hybrid cells injected (Fig. 3), thus indicating that selection for cells with reduced chromosome numbers was occurring in vivo even in populations of hybrid cells which had already undergone some loss of chromosomes in vitro. All tumours showed continued and progressive reduction in chromosome number on repeated transplantation in vivo (Fig. 4). These results clearly indicate that the tumours arising in the animal are produced by selection of cells, but some of the hybrid cells already showing a reduced chromosome number in vitro can give rise to tumours in which the cells do not show a further reduction in chromosome number. o -O E C ) E o E "D 3 indi c ft ~v >g V t> rt C a ti u , j iu,, il -ill. : ijl i. - JL m Ul i ii // i ii Chromosome number in vitro (-2-69). i la tumour (5-2-69) i i i tumour ( ) passage i // i i i tumour (6-3-7) passage 3 i i ill Fig. 4. Progressive loss of chromosomes on continued passage of a tumour arising from a clonal population of Ehrlich/fibroblast hybrids. The initial tumour shows only a slight reduction in modal chromosome number relative to that of the cells injected; subsequent passages show more marked chromosome losses. DISCUSSION These observations on Ehrlich/fibroblast hybrids are, as far as the main point of the investigation is concerned, inconclusive. They do not decide whether a diploid fibroblast fused with a malignant tumour cell can suppress malignancy in the hybrid; but they do show how instability of the chromosomal constitution of the hybrid cell may profoundly affect the interpretation of the results in such fusion experiments. In order to decide whether a normal diploid cell can suppress malignancy, it is obviously necessary to construct hybrids that maintain the complete chromosome sets of the two parent cells at least long enough to permit adequate assays to be made. In the following paper we present some observations on such hybrids.

7 REFERENCES Analysis of malignancy. II 679 FORD, C. E., HAMERTON, J. L., BARNES, D. W. H. & LOUTIT, J. F. (956). Cytological identification of radiation-chimaera8. Nature, Lond. 77, HARRIS, H. (97). Cell Fusion. The Dunham Lectures. Oxford: Clarendon Press. JOHNSON, R. T. & HARRIS, H. (969). DNA synthesis and mitosis in fused cells. II. HeLa-chick erythrocyte heterokaryons. J. Cell Set. 5, JOHNSON, R. T. & RAO, P. N. (97). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature, Lond. 226, JOHNSON, R. T., RAO, P. N. & HUGHES, S. D. (97). Mammalian cell fusion. III. A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species. J. cell. Physiol. 76, SCHWARTZ, A. G., COOK, P. R. & HARRIS, H. (97). Correction of a genetic defect in a mammalian cell. Nature, Lond. (in the Press). (Received 29 September 97) Table. Growth of Ehrlichjfibroblast hybrids in vivo All animals Cell line or clone Ehrlich/T6T6 (i) Ehrlich/T6T6 (2) Ehrlich/T6T6 (3) Ehrlich/T6T6 clonal series Clone Clone 2 Clone 3 Clone 4 Clone 5 Clone 7 Clone 7 B Clone 8 Clone 9 Clone No. takes (Total no. animate with nrnirrpcqi \7 * LJl Utl (Too IVt tumours/total Percen- no. inoculated) tage takes 227/228 7/22 7/8 4/4 27/28 26/27 36/45 / 8/9 /2 26/28 /3 6/ Unirradiated animals Percen- No. takes tage takes 4/ / 68 9/ 82 2/2 6/7 4/4 II/II Irradiated animals No. takes 23/23 85/87 5/7 2/2 27/28 2/2 36/45 II/II 7/8 /2 26/28 /3 6/ Percentage takes IOO 98 IOO 97 IOO 8 IOO Table 2. Chromosomal constitution of Ehrlichlfibroblast hybrids Cell iine Ehrlich tumour Ehrlich/T6T6 () Ehrlich/T6T6 (2) Ehrlich/T6T6 (3) Ljate or examination Total chromosome no. A examined Range Mode: Rangt: Mode IO7 IOO No. bi-armed chromosomes -2 o i-3 A 2 2 3

8 68o U. Bregula, G. Klein and H. Harris Table 3. Chromosomal constitution of clonal populations of Ehrlichlfibroblast hybrids Cell line Date of examination No. cells examined Total chromosome no. Range A Mode Percentage polyploid cells Chromosome no. of polyploid clones Range Mode EhrhchT6T6 (wild type) Clone i Clone 2 Clone 3 Clone 4 Clone 5 Clone 7 B Clone 8 Clone 9 Clone 2. II > 9 > 9 > No mode 84 76