Chromosomal distribution in interspecific in vitro hybrid cells

Q 1968 by Academic Press Inc. Experimental

CHROMOSOMAL DISTRIBUTION IN V1TRO HYBRID R. L. TEPLITZ, Division

379

Cell Research 52, 379-391 (1968)

IN INTERSPECIFIC CELLS

P. E. GUSTAFSON and 0. L. PELLETT

of Cytogenetics, Department

of Pathology,

City of Hope Medical 91010, USA

Center, Duarte,

Calif.

Received December 27, 1967

SEVEKAL

levels of order are involved in organization of chromosome structure and behavior. m’ith respect to the molecular events associated with cell division, it is the deoxyribonucleic acid (DNA) which is of principal concern. In this process, a now familiar sequence is pursued. Semiconservative replication and unwinding of the double helix upon DNA synthesis determines that each metaphase chromatid is a hybrid of old template and newly synthesized DNA strands. Recent experiments have shown that the spatial arrangement of newly synthesized DNA strands [25] and their subsequent distribution to daughter cells [ll, 121 may be highly characteristic and ordered. At another level, the constitution of chromosomes in the interphase nucleus appears so indiscriminate a network of fibrils as to defy the regimentation requisite for mitosis. Nevertheless, it is obvious that some sort of mechanism permits this as well as the complicated pairing between homologs in meiosis. Furthermore, it has been noted that despite the disruptive effects of preparing metaphase spreads, the position of chromosomes is not entirely random [13, 14, 181. Labeling of tissue culture cells with tritiated thymidine results in a reproducible, constant distribution of label within the cell [9]. This implies an order to the initiation of DNA synthesis within the chromosome and a fixed spatial orientation of the chromosome within the cell. Otherwise, a random pattern of label would have resulted. Nevertheless, the extent to which chromosomes may have a fixed interrelationship has not been fully investigated. Whether or not substantial order exists for their distribution upon somatic cell division is also incompletely demonstrated. The experiments reported herein yield data upon these points, particularly the latter. 1 This investigation was supported in part by NIH lowship established in the name of Paul W. Priddy.

grant CA 08791-01 and a Research

Experimental

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MATERIALS

AND

METHODS

Embryonic somatic cells of Nos frrwws and Nrrstrlu z~ision \verr grown irl vilro it1 an atmosphere of 5 per cent CO, and air at 3i”C. I+je’s minimum essential t1letliuni was supplemented with 10 per cent serum, 0.03 per cent glutamine and au tibiotics. Cells were initially obtained from 1’. I<. Basrur (Cniversit?- of Gurlph. Ont.. Chnada). Later. mink (Xleutian strain) were obtained from Blue Granite 1:(II. I~arm. Granite Falls, \Vasb. and cultures prepared from them. 13o\-ine (rantl~~ni bred) 01’ from cells were obtained from primary cultures established in our ow1 laboratop Flow Laboratories, Inglewood, Calif. Cultures were fed twice weekly and transferred when confluent. usualI> once weekly. \Vben hybridization between the cells was to bc induced, the\- xc’rc t r!-llsinized (0.25 per cent buffered trypsin), counted and mixed in equal proport ion. lncubation at 3Y for 2 I1 preceded plating. A final concentration of 2 10” !‘111I of cacl1 cell type was achieved. Although the medium and other factors baw been modified, the cultural procedures arc essentially those of Hasrur I-l]. Cultured cells were l1arvcsted for chromosonial analysis following a -4-h incubation with 0.2 nil 1OP .Tf colcemide/lO ml media. Following a saline (0.9 per cent ) wash at 37’C, cells were trypsinized and concentrated by centrifugation. .I saline wish was repeated and tlien tlie cells were csposed to 0.4 per cent sodium citrale for 10 min, sedimented and fixed in a 3: 1 mixture of methanol: glacial acetic aci(l. (:hromosome spreads were made b>- air dry or flame dry technics 1231.

RESULTS

a diploicl c~omplemenl of Lhc hovi~w (212 =W), all chroniosoii~c4 art’ acrocentric hut the S ant1 Y \\hich are respectively a large and small mctaIn

centric (Fig 1 (I). Mink (2~1 -30) chroinosonws are all mctacrwtricl)uI the The Y is small ant1 it5 c*cnsmallest aulosomal pair, \\-hich is acrocrntric. tronlere location is olten indistinct (Fig. 1 b). This n1ink karyotypts, \\.hich clifTers slightly front that shorn-n by Shida and Sasaki [l iI, incluclt+ a So.

3 and So. 8 \vith prominent secondary constrictions (see Fig. 1 h), nluking them potentially useful as marker chromosomes. Similarly, the mlall mink Y is also useful as a marker. Ho\\-ever, the tliwrgencc hct\vccn these species chromosomes virtually makes of each one a marker. Control rultures consisting of parental cells \vere treatetl in an itlt’ntical manner. No Kohertsonian changes were induced in them 1)~ the Itysincold-shock treatment. Parental cell lines remained unchanged over the course of eight months in culture. Hybrid cells appear I\-ithin mixed cultures during the first \vcek, I)ut constitute only qj-10 per cent of the total. After six weeks, hybrid c*t*lls range from 5OLO5 per cent of the mitotic cells of the culture (see Fig. 2). .\IorphoE.q)erimentccl

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Pig. 1. (a) Karotype of Bos tam-us. The complement consists of 24 pairs of acrocentric autosomes differing in length. The only metacentric elements are the large ?i and the small Y. (h) Karotype of Musfela o&ion. There are 11 pairs of metacentric chromosomes including the minute Y and medium-sized S, with the smallest pair of autosomes as the only acrocentrics. Arrows indicate the prominent pericentromeric constriction of the No. 3 and the even more marked constriction of one arm of the No. 8. The latter appeared in size as either the No. 7 or No. 8 and the discrepancy in the visibility of the constrictions between the homologs is worth noting. It was common lo have this difference and casts uncertainly upon the usefulness of the constriction as a marker in hybrid cells (see text).

R. L. Teplitz, P. E. Gustafson and 0. L. Fellett

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logic distinction between the parental cells is readily achieved, since the bovine cell is epithelioid and the mink, fibroblastic. An intermediate morphology is characteristic of the hybrid, confirmed in similar experiments of Basrur. Estimates of cell populations by morphology have alvvays corresponded to evaluation by chromosomal methods. That these hybrid cells

Fig. 2.-Hybridization of mink and cattle cells in vitro. Each point is the average of three experiments. The range is given at the 6-week period. Some cultures are 95 y0 hybrid cells by that time, while others may have only 45-50 ‘$& Abscissa: Time in culture (weeks); ordinate: oh hybrids. 0

I

2

3

4

5

6

7

8

are not merely fused diploid mink and cattle cells, but have only the haploid complement of each, may be seen in Fig. 3. Union of haploid cells of each species would yield a hybrid with 45 chromosomes, 15 of which would be metacentric and 30 acrocentric. The in vitro hybrid observed is within one or two chromosomes of this pattern with 90-95 per cent frequency. In experiments designed to elaborate the factors enhancing hybridization, a reduction in culture temperature from 37°C (20°C or below) for more than 4 hs was found to reverse the process. Cultures in which 90-95 per cent of the cells vvere hybrid would revert in a single transfer within 72 h almost completely to the parental lines. DISCUSSION

Formation of the hybrid karyotype might occur from bovine cells alone. Robertsonian changes involving 26 acrocentrics could form 13 metacentrics. Together with the sex chromosomes, this would provide 15 metacentrics, and with the remaining acrocentric chromosomes, approximate the hybrid karyotype. However, in bovine cells cultured alone and undergoing trypsinization and cold shock throughout an experimental period of 8 months, not one additional metacentric occurred. Using secondarv constrictions for chromosomal identification may be questionable in hybrid cells, In the mink (as in other) cells, secondary constrictions may vary considerably betvveen homologs (see Fig. 1 b). Thus, a Experimental

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Fig. 3.--Hybrid cell. Metaphase spread and karyotype. A prominent pericentromeric constriction identifies the mink No. 3 (arram) but that of the mink No. 8 is uncertain.The smallest metacentric is consistent with the bovine Y (arrow). There are 46 chromosomes present, near the exact hybrid number of 45. The smallest chromosome is indistinct and is consistent in size with the mink Y.

No. 8 mink chromosome constriction convincingly. [16] at critical phases in constrictions. Many other

in a hybrid cell may fail to show the secondary Roth fixative [ 151 and calcium ion concentration the preparation are known to modify secondary parameters of cell culture or chromosome prepaExperimenful

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ration

may also do so. Kerertheless, the mink No. 3, the Y, an(l~or -Yo. s be identified in many of the hybrid cells. This accollnts f’c,r three mink chromosomes, but these markers in a hybrid cell (often in the wme cell) mitigate against the centric fusion of bovine acrocentric chromosomes as the sole source of the metacentrics of the hybrid cell. Finally, the w\~ersal of the hybrid into diploid parental cells is firm eyidenw that both cell\ [MI’ticipate in the hybrid, for rwersal should produce only bovine cells al~tl no mink, if the hybrid \\-ere derived from bovine alone. Observation of cells immediately after plating (hybridization) sho\\.c*ct frcpent multinucleated cells, but hinucleation \\‘as more common than c~fhcr forms. Estimates of multinucleation exceed 20 per cent. 13inuclcation s~c*c)nclary to fusion is probably the first step in the hybridization process. could

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Also significant is the fact that no endoreduplicated forms were seen in metaphase plates analyzed following cold shock. Temperature changes, particularly cold, are known to enhance endoreduplication [S], but not a single such pattern was exhibited in several thousand cells observed. Chromosomal analyses were performed concomitant with the course of hybridization, beginning 2-1 h post-plating. Although fusion of the two parental cells in the culture precede the appearance of hybrid cells, they could only be seen in the immediate post-cold shock period (Fig. 4). It is concluded from this that reduction from fusion tetraploid cell to a diploid hybrid must occur very rapidly. Ko line of fused cells ever became established in any of these sets of experiments. Hybrid cells with unbalanced genetic contribution from each parent cell presumably resulted from fused cells in which random chromosomal distribution occurred during tetrapolar mitosis. These only appeared early in the hybridization process. At 48-72 h following plating, the population of dividing cells was as follows: parental, 80 per cent: “hybrid”, 5 per cent; “unbalanced hybrid”, 3 per cent, other (i.e. polyploids, etc.), 10 per cent. Those with the unbalanced genotype were either lethal or were selected against, since they disappeared. Thus, while the formation of viable hybrid cells constitute strong evidence for nonrandom movement of chromosomes at anaphase, it does not rule out selection as the operational mechanism for production of hybrid cultures. hIore significant is the reversal phenomenon. If a distributive control did not operate strictly upon the entire haploid complement, a return to diploid parental cell types could not occur as quickly as it did. Hevcrsal experiments might be interpreted as having parental cells remaining in culture (2.5-S per cent of each) repopulating the culture. It is not likely that multiplication of the S-10 per cent parental cells could have overgrown the culture in 72 h. That would require approximately seven or eight divisions for each cell (a total of 27-28 cells derived from each cell in culture), or a mean generation time of 9-10 h instead of the usual 20-24 h. To test this possibility, concentrations of parental cells were plated, ranging from that occurring in the reversal experiment and in multiples of 2 up to 20. The results are seen in Fig. 6. None of these concentrations of parental cells is sufficient to repopulate the culture lvithin the 72-h period. A four-hour period of cold does not change the mean generation time of either parental cell. Furthermore, fusion of cells folio\\-ed by tetrapolar mitosis seems to be the preferred route (Fig. 5). Multipolar mitoses have been seen frequently (10-20 per cent) in early stages of the hybridization procedure, both in this Experimental

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R. L. Teplitz,

P. E. Gustnfson and 0. I,. Pellett

laboratory and by Basrur [4]. For these analyses, the use of colchicine \vas avoided, and observation performed on unstained cells under phase microor some similar process would yield only cells homoscopy. Endomitosis zygous for sex chromosomes (as vvell as the others) in it. Endomitotic tetraplaids, upon tetrapolar division yielding diploid daughter cells, pro(tucc one

b

Fig. 5.

Pig. 6.

Fig. 5.PI+ision-tetraploidy-tctrapolar mitosis (schematic). In this model, recombination is possible because of organization of haploid complements upon adjacent metaphase plates and movement to a common pole determined by the centriole. (a) Bovine cell (acrocentric chromosomes); (b) mink cell (metacentric chromosomes); (c) fusion; (d) DNA synthesis; (e) tetrapolar mitosis; (f) 4 diploid daughter cells,each of which has a haploid set of chromosomes from each parent cell. Fig. B.--Parental cell growth curves. Dotted lines represent cells placed in 100 20 nim petri dishes, and the solid lines 60 x 15 mm petri dishes. The larger were used in the reversal cxperiments. The lowest point at time 0 is 0.25 x 104, the approximate number of parental cells in cuture at the time hybrid cells were “reversed” by cold treatment. Steady increase in number occurs in small plates (higher concentration of cells/medium), but even in these the number of cells at 72 h does not exceed 0.25 x 104. Comparable reversed hybrid cell cultures contained 1 1Oj1 x lo6 cells. Only when plating around 4 x 10” cells was there sufficient growth in the time period (7.2 x 105) to suggest comparable repopulation. Abscissa: h of incubation, 0 --52 11; ordinute: IIUIW ber of cells/plate (in 10,000).

population of cells which would have a sex chromosome constitution of YY. Since this is a lethal combination, cultures would be composed only of XX parental cells. This did not occur. Cultures were XX/XV mixtures as expected from fusion derivatives. A culture once reversed, could be induced to re-form hybrids by repeating the trypsin, cold-shock treatment. The organization of chromosome structure which assures the segregation of DNA strands as described by Lark, and haploid complements as reported herein, is not fully known. Sved [22] has suggested that related phenomExperimental

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ena in tetraploid plants may be due to adjacent telomeric attachments of homologous chromosomes to the nuclear membrane, thus assuring coincident movement in cell division. Many other data similarly point toward a mechanism of this type. The frequent association of homologues at mitotic metaphase [18], the regular peripheral position of several chromosomes in metaphase spreads [2, 13, 141, and the unique characteristics of meiotic pairing indicate that the positions of chromosomes within the nucleus are far from fortuitous. Grell’s evidence for a size-dependent mechanism affecting distribution of chromosome No. 4 in Drosophila melanogaster [7] tends to support this thesis. The small chromosome No. 4 would move with a small fragment of an X-chromosome when translocations and/or duplications were present in the complement. In our view, the translocations and other abnormalities act to destroy the normal homologous relationship. i.e. adjacent telomeric positions at the nuclear membrane. Thus, the No. 4 and the Xfragment are free to associate. Differences between the data on these bovine-mink hybrids and those of other “hybridized” cells are pertinent. In the experiments of Barski, as well as Sorieul and Ephrussi and coworkers, at least one cell of the mating is aneuploid [ 1, 191. Aneuploid cell lines are frequently distinguished by a wide modal chromosome range rather than a single number [20]. Cloning of aneuploid cells often produces a modal range rather than a specific unchanging chromosome number [S]. These data indicate a serious and possible irrevocable disturbance of the mechanisms of chromosome distribution. Precisely this cytopathology may prevent the matings of aneuploid/aneuploid or aneuploid/diploid cells from undergoing the recombination requisite for true hybrid formation. 4n example of this type of hybrid (aneuploid/diploid) in the present system is the result of mating mink cells from plasmacytosis (Aleutian) disease with normal bovine cells. Mink plasmacytosis cells which had been grown continuously in culture were obtained from J. Salk, La Jolla, California. These cells had a modal number of 30 with a range of 28-31 (excluding 10 per cent tetraploids) and were pseudodiploid. Common karyotypes showed unpaired chromosomes, translocation chromosomes and fragments (Fig. 7). When hybrids occurred between these mink cells and the normal bovine, a greater range of chromosome number for the hybridized cell was obtained, from 44-76 (Fig. 8). Although many cells had mixtures of appropriate numbers of chromosomes, there were others which were obviously not composed of a haploid, or near haploid, chromosome complement from each speExperimental

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Fig. i.-Plasmacytosis (mink) cell karyolypr. Although the chromosome mode is 30, unpaired and seemingly broken chromosomes establish this as a pseudodiploid number. Even the mink 5 is uncertain, appearing more acroccntric than metacentric. However, the size suggests thal it may- be the result of pericenlric inversion.

ties. Up to “0 per cent of the cells showed a random mixture of mink and bovine chromosomes. %‘hen reversal of these “hybridized” cultures occurred following incubation at louvered temperatures, the cultures often died. Presumably, the difference between reversal of diploid/diploid and aneuploid/diploid hybrids is due to the abrogation of the control of distribution of chromosomes inherent in the aneuploid mink cell; a pathological fcaturr contributed to the aneuploid mink/bovine hybrid. Thus, the parental cells were not re-formed in the reversal experiment, Sometimes the mink parent line became dominant, but only after sufficient time (several xveeks) hat1 elapsed to permit regrowth by remaining parental cells and not by rwonstitution from hybrids. Genetic alterations in the plasmac$~sis mink cell lint may include loss of control mechanisms affecting chromosome distribution. These may bc virus-mediated, as Aleutian disease is thought to be due to virus [3]. That the ccl1 fusion and recombination is not limited to the i/l roils cnvironment is illustrated by the report ol’ Stone et nl. 121 1 m lvhich a chimcrit bull suddenly sholvetl 96 per ccnl recombinant erythrocgtes insteatl of the original two populations. The bone marroxv cells \vere essentially diploid, evidence against the possibility of fusion tetraploids. Data from studies on fusion of leukemic cells after short-term c’ullurc IXsuggest similar h rhino occurrences in neoplasia. Such a mechanism, followed bp recombination, offers an alternative to double nondis,junction as the E.rperimental

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Fig. 8..-Hybrid karyotype between plasmacytosis mink and cattle cells. It has 4 chromosomes. There are numerous adventitious constrictions, a ring chromosome, translocations and gaps or breaks. Identification of “markers” was vague in such instances.

explanation for the double Philadelphia chromosome (Phi) cell in some cases of chronic granulocytic leukemia [lo, 241. These data indicate the presence of a mechanism which controls the movement of mitotic chromosomes into daughter cells at anaphase, at least in the model of interspecific hybrid cells described in these experiments. This mechanism has been referred to as distribution control, distinguishing it from segregation control, a term usually used with reference to other specitic chromosomal events. Interference with distribution control thus may have a role in oncogenesis. The frequency of aneuploidy or pseudodiploidy in malignant cells suggests this. Data obtained with hybrids in which one parental cell was aneuploid is additional suggestive evidence. If normal cell division is characterized by invariant chromosome allotment and cancer is not, this operational disturbance is of prime importance in oncogenesis. Production of stem lines or a repeated karyotype in malignancy indicates that repair of this mechanism may occur after the stem line number has been developed. Such a thesis offers additional experimental approaches to certain questions in oncology; for example, what mechanisms alter the genome of the 26

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neoplastic cell? Loss of distribution control offers the possibility of ne\v chromosomal contributions to daughter cells upon mitosis. M’hile most of such experiments of Nature would be expected to fail, some are likely to survive and establish a clone which map compete successfully \\ith unchangecl diploid cells. SUMMARY

Cells of t\vo species, mink (Mzzstela u&on) and cattle (Bos tnums) \verc gro\vn in culture. After trypsinization, the cells were mixed in equal proporand plated. Fusion of cells followed within 24 h, but tions, cold-shocked did not persist. Instead, recombination took place following tetrapolar mito& and hybrid mink-cattle cells resulted. These gradually became the doninant cell of the culture. When hybrid cells made up 9.3 per cent of Ihc culture, they xvere again subjected to a decline in temperature. .U this time, they reversed to parental lines \\ithin 72 h. When an aneuploid mink ccl1 \\-as used instead of a normal diploid cell, both processes, hybridization and rarersal, lvere severely curtailed. From these experiments, it has been concluded that in normal ~11s a mechanism (distribution control) strictly regulates morcment of a haploitl set of mitotic chromosomes into daughter cells upon cell division. It is also inferred that oncogenesis involves (primarily or secondarily) loss of this regulatory process. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 11. 15. 16. 17. 18. 19.

BARSKI, G., SCIHIEUL, S. and CORNEFERT, F., Comik Rend. Acad. Sci. 251, 1825 (1960). BARTON, D. E., DAVID, F. N. and MERRINCTTON, il., Ann. Hum. Genet. 29, 139 (1965). BASRUR, P. Ii., GRAY, D. P. and KARSTAD, L., Canad. J. Camp. Med. 27, 301 (1963). BASRUR, P. I<., LTnpublished data (1966). BLACK, P. H., ROWE, W. P. and COOPER, H. L., Proc. Nat1 Acad. Sci. L’S 50, 847 (1963). CERNY, M., BAUOYSOVA, M. and HOLECKOVA, E., Exptl Cell Res. 40, 673 (1965). GRELL, R. F., Proc. Nat1 Acad. Sci. lJS 52, 226 (1964). HAINES, M., Nature (London) 207, 552 (1965). HARRIS, H., Biochem. J. 72, 54 (1959). KIOSSOGLO~, K. A., MITUS, W. J. and DAYESHEK, W., Lancet 2, 665 (1965). LARK, K. G. and BIRD, R. E., Proc. Nail Acad. Sci. US 54, 1444 (1965). LARK, K. G., CONSIGLI, R. A. and MINOCHA, H. C., Science 154, 1202 (1966). MILLER, 0. J., MUKHERJEE, B. B., BREG, W. R. and GAMBLE, A. V. N., Cytogenetics 2, 1 (1963). MILLER, 0. J., MUKHERJEE, B. B., GAMBLE, A. V. N. and CHRISTAKOS, A. C., Cyfogenetics 2, 152 (1963). SAKSELA, E. and MOORHEAD, P. S., Cytogenetics 1, 225 (1962). SASAKI, M. and MAKINO, S., Am. J. Hum. Genet. 15, 24 (1963). SHIDA, G. and SASAKI, M., Dobufsugaku Zasshi (Zool. Mag.) 71, 98 (1962). SCHEIDERMAN, L. and SMITH, C. A. B., Nature (London) 195, 1229 (1962). SORIEUL, S. and EPHRUSSI, B., Nature (London) 190, 653 (1961).

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SPUIINA, V. and HILL, M., Neoplasma 14, 11 (1967). STOSE, 1%‘. H., FRIEDMAN, J. and FREGIX, A., Proc. NalI Acad. Sci. US 51, 1036 (1964). 22. SVED, J. A., Genetics 53, 747 (1966). 23. TEPLITZ, R. L., HANSSON, K. M., FOLEY, V. L. and TEPLITZ, hI. R., Eq~tl Cell Res. 41, 686 (1966). 21. TEPLITZ, R. L., in G. D. AnrRoMIN (ed.), Pathology of Leukemia, p. 162. Hoeber Medical Division, 1968. 25. W’ALES, K. H., Genetics 51, 91.5 (1965).

20. 21.

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