537

NOTES

Potter, M., Nadeau, J.H., and Cancro, M.P. (Editors). 1986. The wild mouse in Immunology. Springer-Verlag, Berlin. Sage, R.D. 1981. Wild mice. In The mouse in biomedical research. Vol. 1. Edited by H.L. Foster, J.D. Small, and J.G. Fox. Academic Press, New York. pp. 39-90.

Tucker, P.K., Lee, B.K., and Eicher, E.M. 1989. Y chromosome evolution in the subgenus Mus (genus Mus). Genetics, 122: 169-179.

Chron~osomesegregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors Genome Downloaded from www.nrcresearchpress.com by STANFORD UNIV. on 11/25/14 For personal use only.

JENNIFER A. MARSHALL GRAVES'

AND

IOLE BARBIERI

Department of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia

Corresponding Editor: R. Appels Received December 14, 1991 Accepted December 24, 1991 GRAVES, J. A. M., and BARBIERI, I. 1992. Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors. Genome, 35: 537-540. Using human and Chinese hamster established lines as cell parents, we constructed hamster-human cell hybrids and human cell - hamster karyoplast hybrids. The cell hybrids retained one or two sets of hamster chromosomes and lost most of the human chromosomes. The karyoplast hybrids, however, retained a full set of human chromosomes and lost most of the Chinese hamster chromosomes. This reverse segregation pattern implies that cytoplasmic factors are major determinants of the direction of chromosome segregation. Key words: cell hybrids, chromosome loss, cytoplasmic factors, reverse segregation. GRAVES, J. A. M., et BARBIERI, I. 1992. Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors. Genome, 35 : 537-540. Le recours a des lignees etablies de cellules humaines et de hamsters, comme cellules parentales, a permis de produire Chez des hybrides de cellules hamster-humain >> et des hybrides cellules humaines - caryoplastes de hamsters >>. les cellules hybrides, une ou deux series des chromosomes de hamster ont ete retenues et la majorit6 des chromosomes humains ont etC perdus. Chez les caryoplastes hybrides, une serie complete des chromosomes humains ont kt6 retenus, mais la plupart des chromosomes de hamster ont ete perdus. Ce patron de segregation inversee sous-tend que des facteurs cytoplasmiques seraient les determinants majeurs de la direction de la segregation des chromosomes. Mots elks : cellules hybrides, perte de chromosomes, facteurs cytoplasmiques, segregation inversee. [Traduit par la redaction]

Introduction Although chromosome segregation from mammalian cell hybrids is a crucial element of somatic cell genetic analysis, its mechanism still remains mysterious. The direction and extent of chromosome loss vary greatly according t o the combination of parental cells fused, and it has been possible to construct a set of rules of thumb, even in the absence of any real understanding of the mechanism of segregation. Interspecific hybrids normally retain an intact chromosome set from one parental species (the "retained set") and eliminate chromosomes preferentially from the other (the "segregant set"). Most notably, the direction of segregation seems to depend on the species of origin of the parental cells and is rather difficult to reverse. For instance, rodenthuman hybrids almost invariably segregate human chromosomes; loss is rapid and progressive (Weiss and Green 1967). Reversal of this direction has been reported in hybrids in which established human cells were fused with diploid rodent cells, suggesting an overriding effect of cell transformation (Minna and Coon 1974; Croce 1976). However, reversal of the direction of loss was also accomplished by varying the input ratio of parental components in experiments with polyploid series of mouse and hamster Printed in Canada / Imprime au Canada

cells (Graves 1984), so the effect of cell transformation may be, after all, a dosage effect. The dosage effect on the direction and extent of segregation was originally ascribed to differences in dosage of particular nuclear genes (Graves 1984). However, polyploid cells will contribute more cytoplasmic, as well as nuclear, factors, in proportion to their ploidy. Thus, it is possible that the reversal of chr~mosomesegregation in hybrids made between cells of different ploidy is a function of dosage differences, not in nuclear genes, but in some cytoplasmic component. It is possible to test the hypothesis that the cytoplasm contains factors that direct chromosome loss by constructing hybrids from karyoplasts, cell membrane bounded "minicells" containing a nucleus surrounded by little or no cytoplasm, and comparing the direction and extent of segregation with hybrids constructed from whole cells of the same parental lines. We have compared the chromosome segregation from Chinese hamster - human hybrids with segregation from hamster karyoplast - human cell hybrids and report here that the direction of chromosome segregation is reversed, implying that cytoplasmic rather than nuclear factors set the direction of chromosome loss.

538

GENOME, VOL. 35, 1992

TABLE1. Chromosome constitution of intact cell and karyoplast hybrids

Genome Downloaded from www.nrcresearchpress.com by STANFORD UNIV. on 11/25/14 For personal use only.

Modal numbers of chromosomes Cell lines

Hamster

Human

V79 hamster HBU human V79 x HBU cell hybrids (n = 26) V79 karyoplast x HBU nuclear hybrids (n = 12)

21

-

-

115

20

4-12

1-15*

109

*Some hybrids retained only fragments of hamster chromosomes translocated to human chromosomes.

Materials and methods Parent cell lines and cell culture The Chinese hamster parent line V79-129 is a hypoxanthine phosphoribosyltransferase (HPRT) deficient derivative of V79 obtained from M. Harris, Berkeley (Harris 1972). The human cell parent was HBU, a thymidine kinase (TK) deficient HeLa derivative (Kit et al. 1966) obtained from E. Stanbridge, Stanford. Cells were routinely cultured in Dulbecco's modification of Eagle's medium (DME, Flow, Australia) supplemented by 10% foetal calf serum (Flow), 60 pg/mL penicillin (Sigma), 50 pg/mL streptomycin (Glaxc, Australia), and 100 pg/mL glutamine (Calbiochem). Karyoplast preparation Cells were enucleated by a modification of the methods first described by Ege et al. (1974) and Wigler and Weinstein (1975). At least 60 x lo6 cells were harvested and resuspended at 0.5-2 x lo7 cells/mL in 12.5% Ficoll in DME containing 10 pg/mL Cytochalasin B (Sigma) and 0.5 % DMSO. Three millilitres of the cell suspension were layered onto a Ficoll gradient (12.5-25% Ficoll in DME at pH 7.0-7.4), which had been incubated overnight at 37"C, and 3 mL medium containing 10 pg/mL Cytochalasin B was overlayed before centrifuging for 60 min at 25 000 rpm at 31°C in a prewarmed rotor. Bands were identified under strong illumination and were carefully removed one by one using Pasteur pipettes. A small drop from each was sampled, fixed, spread onto a microscope slide, and stained with Giemsa; the proportions of whole cells, enucleate cells (cytoplasts), and karyoplasts were determined for each. Cell and karyoplast hybridization To obtain hybrids between whole cells, V79 and HBU cells were fused in a 1:1 ratio in suspension using polyethylene glycol (MW = 1000, Ajax, Australia) diluted to a 50% w/v solution in serumfree DME. Fused cells were cultured overnight in 100-mm Petri dishes, then changed to HAT medium (Szybalski et al. 1962) conaminopterin, and 1.6 x taining 10 - 4 M hypoxanthine, 4 x M thymidine (Sigma) in a humidified CO, incubator. After 2-5 weeks of growth, individual colonies were separated using glass cloning rings. To obtain hybrids between hamster karyoplasts and whole human cells, karyoplasts were prepared from V79-129 cells, their purity was tested as described above, and they were counted, mixed with an equal number of HBU cells, and fused as above. Only the preparations that produced a sufficient yield (5-10 x lo6) of pure karyoplasts were fused. Chromosome studies Hybrid cells were harvested, swelled 15 min in 0.75 M KC1, then fixed in 3:l methanol - acetic acid and left overnight before airdried preparations were made. Hamster and human chromosomes could be easily distinguished by a modification of the G-1 1 banding technique (Bobrow and Cross 1974) in which slides were placed

in 2 x SSC at 58-62°C for 5 min, rinsed in double-distilled water, incubated in 4% Giemsa (Harleco, Lab Aids, Sydney) in 0.01 M NaOH at 37°C for 9 min, rinsed, and air dried. Cells were examined using a Leitz Dialux 2 microscope and photographed using Kodachrome slide film or Kodak technical pan.

Results Cell hybrids Colonies were recovered from fusions between whole hamster and human cells at frequencies of 10 - '-10 - in HAT medium. Challenge with M ouabain (which is toxic to human but not hamster cells) eliminated most of these colonies, suggesting that they were TK revertants of the human line (which has a reversion frequency in excess of 10 -6). The remaining colonies were propagated in HAT with ouabain until they could be karyotyped. Hybrids constructed between V79 and HBU intact cells clearly retained one (occasionally two) set@) of hamster chromosomes and segregated most of the human chromosomes, retaining only up to 12 of the originally 115 human chromosomes of the HBU line (Table 1, Fig. la). +

Karyoplast hybrids Of many Chinese hamster and human cell lines tested, the one that gave the purest karyoplast preparations was the V79-129 Chinese hamster line. Colonies were isolated from fusions between hamster karyoplasts and human cells at frequencies comparable to those for cell hybrids (near but again, most appeared to be ouabain-sensitive human revertants, containing no cytologically identifiable hamster chromosome material. Those that survived ouabain selection were karyotyped using G-1 1 banding. In contrast with the cell hybrids, hybrids constructed between hamster karyoplasts and intact human cells retained one (occasionally two) complement(s) of human HBU chromosomes and segregated hamster chromosomes (Table 1, Fig. lb). One karyoplast hybrid (VkH 38) examined in detail retained 8-12 hamster chromosomes, easily identified by GI1 banding (Fig. lb), and another contained one small hamster chromosome and a hamsterhuman translocation chromosome. Several others retained only fragments of hamster chromosomes (identified by their magenta colour) translocated to a human chromosome. Four hybrids had several magenta-stained double minutes, the only evidence of a hamster chromosome contribution.

Discussion Despite its importance for human gene mapping, the mechanism of preferential chromosome segregation from interspecific cell hybrids is still unknown. An effect of dosage of the parental contributions on the direction and extent of chromosome segregation has been clearly demonstrated (Graves 1984). Experiments of Pravtcheva and Ruddle (1982, 1983) suggested that an altered dosage of individual chromosomes (specifically the mouse X chromosome) could influence segregation, even causing reversal of the direction of segregation. Thus, the dosage effect has generally been ascribed to dosage of a particular gene or genes. The present experiments contradict this assumption, for they demonstrate very clearly that the direction of chromosome loss is reversed when the relative proportions of hamster and human cytoplasm is altered, while the proportion of nuclei is kept constant. In hybrids made between

Genome Downloaded from www.nrcresearchpress.com by STANFORD UNIV. on 11/25/14 For personal use only.

NOTES

FIG. 1. Reverse chromosome segregation from hybrids between hamster karyoplasts and intact human cells. (a) G11-stained chromosomes of a Chinese hamster x human cell hybrid constructed by fusion of intact V79 and HBU cells. There are 43 magenta-stained hamster chromosomes (darker in this print) and 6 blue-stained (lighter) human chromosomes. (b) G11-stained chromosomes of a Chinese hamster x human nuclear hybrid constructed by fusing V79 karyoplasts with intact HBU cells. There are 127 blue (lighter) human chromosomes and 15 magenta (darker) hamster chromosomes, as well as a number of double minutes.

Genome Downloaded from www.nrcresearchpress.com by STANFORD UNIV. on 11/25/14 For personal use only.

540

GENOME, VOL. 35, 1992

whole V79 Chinese hamster and HBU human cells, human chromosomes were rapidly and almost completely lost, as is the case for all of the many other hamster-human cell hybrids reported previously. However, when hybrids were made between V79 hamster karyoplasts and the same HBU human cell line, chromosome loss was reversed. The cell hybrids and karyoplast hybrids each received one (or sometimes two) hamster and one human nucleus: the only difference between the input into these hybrids was the absence of hamster cytoplasm from the karyoplast hybrids. Thus reverse chromosome segregation in these hybrids must depend at least in part on the presence of cytoplasmic factors. Heterokaryons and hybrids between karyoplasts and intact cells have been used previously to investigate the nuclear and cytoplasmic factors controlling cell aging (Muggleton-Harris and Palumbo 1979), differentiation (McBurney and Strutt 1979), and hybrid viability (Weide et al. 1982). The last two of these reports contain some data on chromosome numbers in intraspecific (mouse-mouse) nuclear hybrids. McBurney and Strutt (1979) observed no difference between the chromosome numbers of hybrids and nuclear hybrids, whereas Weide et al. (1982) observed a decrease, which they attributed to the more rapid growth (and thus increased number of divisions) of the latter. No data are available on .the chromosome constitution of hybrids between rodent karyoplasts and intact human cells. Reversal of the direction of chromosome segregation in karyoplast hybrids suggests that there are factors in hamster cytoplasm that have an overriding effect over factors in human cytoplasm in promoting the retention of specieshomologous chromosomes. In the absence (or great proportional reduction) of hamster cytoplasm, factors in the human cytoplasm promote the retention of human and the segregation of hamster chromosomes. Our previous observation of a dose-dependent influence of parental contributions on the direction and extent of chromosome segregation from mouse-hamster hybrids (Graves 1984) can be accounted for by the same hypothesis, although the action, in addition, of dosage-dependent nuclear factors cannot be excluded and is suggested by the results of Pravtcheva and Ruddle (1982, 1983). We conclude, therefore, that the cytoplasm contains molecules or structures that are involved with chromosome segregation and that act in a species-specific and competitive fashion in interspecific hybrids. The identity of these factors remains unknown: they could be constituents of autonomous self-replicating organelles (e.g., mitochondria1 components) or cytoplasmic structures (e.g., components of the mitotic apparatus) that owe their origin ultimately to nuclear gene activity. Direction of loss has previously been shown to be independent of parental contributions of 0-tubulin (Zelesco and Graves 1987), but the roles of other spindle components are unknown. The identification of these cytoplasmic factors, and investigation of their mode of action, is likely to provide new information on the normal

process of chromosome segregation at mitosis in mammalian cells. Acknowledgements This work was supported by a grant to Dr. Graves from the Australian National Health and Medical Research Council. We thank Maria Tarzia for assistance with the preparation of the manuscript. Bobrow, M., and Cross, J. 1974. Differential staining of human and mouse chromosomes in interspecific cell hybrids. Nature (London), 251: 77-79. Croce, C.M. 1976. Loss of mouse chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritoneal macrophages. Proc. Natl. Acad. Sci. U.S.A. 73: 3248-3252. Ege, T., Hamberg, H., Krondahl, U., et al. 1974. Characterization of minicells (nuclei) obtained by cytochalasin enucleation. Exp. Cell Res. 87: 365-377. Graves, J.A.M. 1984. Chromosome segregation from cell hybrids. I. The effect of parent cell ploidy on segregation from mouse - Chinese hamster hybrids. Can. J. Genet. Cytol. 26: 557-563. Harris, M. 1972. Effect of X-irradiation of one partner on hybrid frequency in fusions between Chinese hamster cells. J. Cell. Physiol. 80: 1 19-128. Kit, S., Dubbs, D.R., and Frearson, P.M. 1966. HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity. Int. J. Cancer, 1: 19-30. McBurney, M.W., and Strutt, B. 1979. Fusion of embryonal carcinoma cells to fibroblast cells, cytoplasts and karyoplasts. Exp. Cell Res. 124: 17 1-180. Minna, J.D., and Coon, H.G. 1974. Human x mouse hybrid cells segregating mouse chromosomes and isozyrnes. Nature (London), 252: 40 1 -404. Muggleton-Harris, A.L., and Palumbo, M. 1979. Nucleocytoplasmic interactions in experimental binucleates formed from normal and transformed components. Somatic Cell Genet. 5: 397-407. Pravtcheva, D.D., and Ruddle, F.H. 1982. X chromosome control of chromosome segregation in mouse/hamster hybrid cell populations. Ann. N.Y. Acad. Sci. 397: 249-255. Pravtcheva, D.D., and Ruddle, F.H. 1983. X chromosome-induced reversion of chromosome segregation in mouse/Chinese hamster somatic cell hybrids. Exp. Cell Res. 146: 401-416. Szybalski, W., Szybalska, E.H., and Ragni, G. 1962. Genetic studies with human cell lines. Natl. Cancer Inst. Monogr. 7: 75-89. Weide, L.G., Clark, M.A., Rupert, C.S., and Shay, J.W. 1982. Detrimental effect of mitochondria on hybrid cell survival. Somatic Cell Genet. 8: 15-21. Weiss, M.C., and Green, H. 1967. Human-mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl. Acad. Sci. U.S.A. 58: 1104-1111. Wigler, M.H., and Weinstein, I.B. 1975. A preparative method for obtaining enucleate mammalian cells. Biochim. Biophys. Res. Comm. 63: 669-673. Zelesco, P .A., and Graves, J .A.M. 1987. Chromosome segregation from cell hybrids. 111. Segregation is independent of spindle constitution. Genome, 29: 528-53 1 .

Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors.

Using human and Chinese hamster established lines as cell parents, we constructed hamster-human cell hybrids and human cell - hamster karyoplast hybri...
343KB Sizes 0 Downloads 0 Views