Phenotypes of cell hybrids formed by fusion of normal and Rous sarcoma virus transformed cells

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PHENOTYPES OF CELL HYBRIDS FORMED BY FUSION OF NORMAL AND ROUS SARCOMA VIRUS TRANSFORMED CELLS. Thorfinn Ege Department of Medical Cell Genetics Medical Nobel Institue, Karolinska S-10401 Stockholm 60, Sweden and Department of Biochemistry Norsk Hydra's Institute for Cancer Det Norske Radiumhospital Montebello, Oslo 3, Norway

Institutet

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SUMMARY Untransformed mouse cells were fused with rat cells transformed by a temperature sensitive mutant of avian sarcoma virus, and cell hybrids were isolated in the absence and in the presence of selective medium. None of the hybrids isolated were as transformed as the parent rat cells. All hybrids isolated in the absence of selective medium showed a phenotype similar to that of the untransformed mouse cell parent. Cell hybrids isolated on selective media, however, were more heterogenous. Some showed a phenotype that were intermediate between that of the two parental cells, while others were more like the untransformed mouse cells. INTRODUCTION Cell hybrids have been used by a number of investigators to analyze the genetics of the malignant and transformed phenotype (for a review, see Ozer and Jha 1977). Both supression and dominanse of malignant as well as transformed phenotypes have been observed in hybrids after fusion of malignant or transformed cells with untransformed cells. However, the lesions in the transformed/ malignant partner in such hybrids have often been ill defined. A number of experiments have been performed with virus transformed cell lines, but these lines have often been mutagenized to produce enzyme deficient mutants after the transforming event, or have been carried in culture for extended periods of time, and it is therefore possible that these cells have undergone secondary transformation events. Lack of supression of the transformed phenotype in hybrids involving this type of cells as one of the parents, therefore does not 0309-1651/84/070607-07/$03.0010

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allow the conclusion that virus transformed cells show a dominant expression of the transformed phenotype in cell hybrids. The frequency of cell hybrids that can be isolated is usually orders of magnitude lower than the frequency of heterokaryons present after fusion. As it has been shown (Ege 1984) that most of the heterokaryons are able to give rise to growing daughter cells in the absence of selective pressure, this means that the hybrids normally isolated only represent a small fraction of all cell hybrids formed. It is therefore possible that the isolation procedure normally employed selects for a sub-group of cell hybrids that are not representative for the large mass of hybrids formed. To avoid the above arguments rat cells transformed with a ts mutant of avian sarcoma virus have been used as the transformed cell in cell fusion experiments with normal 3T3-cells. These cells show a tegperature sensitive transformed phenotype. At 35 C the cells grow to high cell densities and in the absence of solid support, while at 39OC the cells are indistinguishable from their untransformed parent (Brzeski and Ege, 1980). It is therefore possible to control if the transformed phenotype of a hybrid cell is caused by the temperature sensitive avian sarcoma virus, or whether it is due to secondary transformation events that have taken place during culture of the hybrid cells. Furthermore, cell hybrids have been isolated under conditions that allow isolation of growing cell hybrids from up to 50% of the heterokaryons (Ege 1984), thereby avoiding the selection of a small fraction of all cell hybrids formed. MATERIALS AND METHODS Cells and culture conditions: Hypoxantine phosphoribosyl transferase deficient normal rat kidney cells, NRK TG', were transformed with a temperature sensitive mutant o'f an avian sarcoma virus (La 3341, and a cell line NRK TG'-La 334, showing a high plating efficient in semi-solid medium, was isolated. NRK TGry-La 334 and 3T3 TK- cells, a thymidine kinase deficient subline of mouse 3T3 cells, were cultivated in Dulbecco's modified Eagles Minimum Essential Medium containing 10% heat inactivated calf serum (normal growth medium). The procedures for fusion, isolation of growing hybrids, and chromosome analysis were as described in the accompanying paper (Ege 1984). Growth in semisolid medium: Parental and hybrid

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cells were tested for growth in semisolid medium b,~( seeding 1000 or 10 000 cells per ml in normal growth 1.3% Methocel. The number of medium containing growing colonies were scored after two weeks. the method of Virus resque: For virus resque, Steiner and Boettiger (1977) was used. Clones of cells to be tested were treated with Mitomycin C (10 2hrs). After overnight incubation in normal q/ml, growth medium, chick fibroblasts were added and the cells fused with polyethylene glycol. Incubation was continued for one week at 35OC before the cultures were scored for the presence of transformed chicken cells.

RESULTS Cell fusion and isolation of growing cells. As previous results have shown that a large number of the hybrid cells formed from binucleated cells are unable to survive HAT selection (Ege 19841, growing hybrid cells were isolated both in the presence and in the absence of selective medium. In cultures where cells were grown in the absence of selective medium, binucleated cells were identified soon after fusion, and surrounded by cloning rings. 2-3 weeks after fusion, colonies of cells surrounded by cloning rings were trypsinized, and the colonies expanded in separate dishes. The percentage of binucleated cells giving rise to growing colonies varied a little from experiment to experiment, but was usually about 40%. Frcm two experiments, 24 cell lines were isolated under non-selective conditions. These were identified as either hybrid cells (16) or tetraploid parental cells based on the LDH isoenzyme pattern and chromosome analysis. All hybrids expressed both parental isoenzymes. From the same two experiments 5 cell lines were isolated after HAT selection. In this -5ase the frequency of growing cells were about 1x10 . All of these lines were identified as hybrids, based on their chromosome constitution. Growth characteristics of hybrids and control cells. The properties of parental and fused cells are shown in tab&e 1. When tested for growth in semisolid medium at 35°C‘ the virustransformed rat cells showed a relatively high plating efficiency with large colonies appearing after one week. Under the same conditions untransformed 3T3TK- mouse cells failed to divide more than once or twice even after

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cell

Clone

type

Crcmosane nr, (range)

Reports, Vol. 8, No. 7, July 1984

Plating efficiency inMethoce1

Virus rescue

NRIzTG:-La 334 3T3TK--

70 i65-83j

z.01

NRKTGr La tetraploid

74 77 78 77

(69-86) (75-81) (72-80) (68-79)

27 0.1 15 4

++ ++ ND ND

128 137 129 134

(81-148) (122-150) (111-136) (119-145)

co.01 (0.01 (0.01 CO.01

ND NE ND

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

107 95 103 108 116 98 108 100 106 95 99 107 100 104 93 100

(98-112) (91-104) (loo-111) (99-115) (100-120) (89-100) (102-112) (89-120) (99-109) (86-105) (91-108) (91-115) (98-107) (100-112) (88-101) (94-115)

(0.01
1s

103 106 98 104 112

(92-109) (101-108) (89-104 (95-109) (93-120)

14 (0.01

40

334

3T3TKtetraploid

N-RK!rGrLa 334 x 3T3TKnon-selected

NIUTd- La x 3T3TKselected

334

2s 3s 4s

5s

(38-42)

2-3

0.2

10 0.1

++k

+ +I+ + + + + + + + + + + + + + i-k + +

Table 1. Properties of parental cells, tetraploid parental cells, and cell hybrids isolated in the absence and presence of selective media. two weeks. Tetraploid 3T3TK- cells were also unable to grow in Methocel while tetraploid NRK TG'-La 334 showed a reduced plating efficiency compared to the diploid cells: Clone 2 cells form very few colonies, and although cells from the other three clones form colonies, they do so at considerably slower rate than the diploid parental cells. 13 out of the 16 hybrids isolated under non-

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as dependant on sol-id selective conditions were support for their growth as were the untransformed parental cells, while three clones showed a slightly The colonies forming increased plating efficiency. from clone 3 and clone 10 cells grew slowly, while the Sew colonies arising from clone 11 grew as fast as the transformed parent cells. Compared to the non-selected hybrids, the ones selected on HAT medium were more heterogenous: Clone 2s cells were as dependent on solid support for growth as were the untransformed parent cells. Clone 3s and 5s showed slightly elavated plating efficiencies compared to the 3T3 parent, but colonies formed slowly and never grew to the same sizes as those from the transformed parent. Two clones (1s and 4s) gave plating efficiencies in Methocel that were somewhat lower than that of the transformed parental cells. When the clones able to grow in Methocel at 35OC were tested at 39OC, only ClOr;f? 1 gave rise tc colonies. None of the parental cells were able to grow at the restricted temperature. Virus could be rescued from most of the hybrid cells, although less efficiently than from the virus transformed parental cell. There was no correlation between the cloning efficiency in semi-solid medium and the efficiency of virus rescue from the different hybrid clones. DISCUSSION When untransformed mouse cells were fused with virustransformed rat cells, all but one of the hybrid clones isolated in the absence of selection were unable to grow in semisolid medium. This indicates that with respect to this parameter the hybrids have a normal phenotype. These hybrid clones were also morphologically different their from transformed parent cells in that they were epitheloid and showed firm attachment to the culture substrate even at high cell densities, conditions that caused the transformed rat cells to round up and become semiattached. Clone 11 cells, which showed an increased plating efficiency in semi-solid medium compared to the untransformed parent cells, grew to higher densities than the rest of the unselected hybrid colonies. However, it did not show the typical round morphology of the transformed rat cells, and other avian sarcoma virus transformed cells, when the cultures reached high densities. Clone 11 cells analysed at 39OC showed the same phenotype as at 35Oc, indicating that it is not the temperature

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sensitive avian sarcoma virus that is responsible for the transrormed phenotype. We interpret this result to mean that clone 11 cells probably have undergone a spontaneous transformation this is and that responsible for the transformed behaviour of the cells. Hybrid clones isolated on HAT medium were more heterogenous than these isolated in the absence of selection. 3 out of 5 clones showed growth characteristics in semisolid medium similar to that of untransformed cells. However, two.clones (IS and 4s) showed efficiency in a relative high plating semisolid medium at 35OC, but not at 39OC, and also morphologically resemled the parental rat cells. This indicates that while the avian sarcoma virus indused transformation was recessive in most of the hybrid cells, the transformed phenotype was expressed in two hybrid clones. It is interesting to note that the two clones showing expression of the transformed phenotype are found alr.ong the five clones isolated from HAT medium, while all the clones isolated in the absence of selection showed dominance of the normal phenotype. As only a fraction of all hybrid cells present survived FiAT selection, it is tempting to speculate that HAT medium not only selects for cell hybrids, but also selects for the most vigorously growing cells, a character often assosiated with the transformed phenotype. Two clones of tetraploid rat cells showed a reduced plating efficiency in semisolid medium compared with the parent rat cells. The reason for this is not understood, but may be due to elimination or amplification of certain groups of chromosomes in the tetraploid cells although no consistent pattern is evident from chromosome of the 4 clones. analysis Although chromosome analysis of the isolated cell hybrids show that chromosome elimination have taken place during their isolation, the recessive behaviour of the transformed phenotype in the above studied hybrid cells can not be explained by the loss of the transforming agent from the hybrids, as a transforming virus can be rescued from most of the hybrid cells. A recessive behaviour of the transformed/malignant phenotype has previously been observed in hybrid cells from crosses between normal and transformed cells (Rarski, 1961, Weiss et al., 1968), and even in crosses between two different transformed cells (Harris, 1971). If the transformed phenotype of the parental cells in cases these were due to gene the normal phenotype of the hybrids could be defects, explained by gene complemention of the two defects.

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As the transformed phenotypt: of RSV transformed cells is caused by the presence of a viral protein in the cells (Purchio et al., 19781, the growth characteristics of the hybr-11s observed in the present study can not be due to such a gene complementation effect. The conclusions obtained in this study confirm previous experiments obtained by Xarshall (1980) who also observed a normal behaviour of hybrid cells made between virus transformed and normal cells. E'urthermore, present results show that different the isolation procedures used for hybrid isolation select populations of cell hybrids with different properties. Acknowledgement: Mariann Frostvik The study was Cancer Society.

The author wish to for skillful1 technical supported by grants from

REFERENCES

thank

.pIiss

assistance.

the

Swedish

:

T (1984) Analysis of heterokaryons and progeny cell hybrids isolated in the absence and in the presence of selective media. Submitted for publication. Harris H (1971) Cell fu sion and the analysis of malignancy. Proc Royal Sot London, Ser B 179-l-20 Marshall CJ (1980) Supression of the transformed phenotype with retention of the viral 'src' gene in cell hybrids between ROUS sarcoma virus transformed rat cells and untransformed mouse cells. Experimental Cell Research 127.373-384 Ozer HL, and Jha KK (1977) Malignancy and transformation: Expression in somatic cell hybrids and variants. Advances in Cancer Research 25.53-93 Purchio AF, Erikson E, Brugge J, and Erikson RL Identification of a polypeptide encoded by (1978). the avian sarcoma virus src gene. Proceedings of the National Academy of Science (USA) 75.1567-1571 Steiner KS and Boettiger D (1977). Complementation rescue of Rous sarcoma virus from transformed mammalian cells by polyethylene glycol-mediated cell fusion. Journal of Virology 23.133-141 Weiss M, Todaro GJ, and Green H (1968) Properties of a hybrid line between lines sensitive and insensitive to contact inhibition of cell division. J.Cell Physiology 17.105WeI

Received:

4th June 1984.

Accepted:

11th

June 1984.