The quantitative distribution pattern of deoxyribonucleic acid in cell cultures

Q 1966 by Academic Press Inc. Experimental

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Cell Research 44, 521-526 (1966)

THE QUANTITATIVE DISTRIBUTION PATTERN DEOXYRIBONUCLEIC ACID IN CELL CULTURES SHEILA Department

M. SPARSHOTT’and of Pathology,

University

OF

E. S. MEEK of Bristol,

England

Received June 20, 1966

WUH

the widespread use of cell cultures in histological studies, it has become apparent that these can be divided into two classes. On the one hand, there are the cultures which, from the time of primary explantation, grow well for a while but later become progressively more feeble; these die out within a year-often much sooner. These are known as cell “strains”. On the other hand, there are those which undergo some form of conversion (often referred to as transformation) resulting in the emergence of cells capable of vigorous growth over an indefinite period; these are the cell “lines”, many of which have been subcultured repeatedly over many years. A notable feature of cell lines is that, unlike cell strains which remain diploid, they sho\v differences in chromosome number from that of the original parent tissue; moreover, there is characteristically a wide range of chromosome numbers present [4]. The similarity in behaviour between cell lines and malignant cells in uivo is striking, and Hayflick [4] considers cell lines to be the in vitro expression of tumour cells. In parallel with the wide range of karyotypes seen in cell lines, there is an equally wide distribution of quantitative values of deoxyribonucleic acid (DNA) present in the mitotic figures. Recent study of a line of rabbit kidney cells (RK13) shows, however, that this wide range of DNA values in mitotic figures, which presumably reflect an equally broad dispersion of genotypes, was not present. MATERIALS

AND METHODS

RK13 cells, (GL-RK13 cells; obtained through the courtesy of Dr G. Christofinis and Dr L. W. Greenham) originally from normal rabbit kidney [l], were grown in Tissue Culture Medium 199 (Glaxo) with 5 per cent calf serum (inactivated by heating at 56°C for 30 min). The cells were grown on flying coverslips in test tubes, and the coverslips then removed, rinsed briefly in physiological saline and fixed in 1 Present address; Department 34- 661810

of Pathology,

Medical

School, Liverpool

3, England.

Experimental

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Sheila 31. Sparshott and E. S. Meek

absolute methyl alcohol for a minimum of 15 min. The preparations were stained by the Feulgen method and quantitative estimations made of the content of DNA in single nuclei using a commercial version of the scanning and integrating microdensitometer designed by Deeley [a]. Control smears of cells were made from fresh normal rabbit kidney. RESULTS The estimations of DNA are given in arbitrary units; these are presented in the accompanying figures and show three significant features. The distribution of values found in cells during interphase is substitute scattered between two limits such that the upper value is twice that of the lower value. This is the range of distribution seen in both normal diploid cell populations in viva and in cell strains (not lines) in uitro. Within such a range, extending from diploid to tetraploid values, the relative distribution depends on the rate of cell division. In a non-dividing population, such as that of small lymphocytes, there is a narrow peak at the diploid value only. With a high rate of division, values are found up to the tetraploid level which is seen in cells about to enter mitosis. The present findings resemble those of a diploid population with a very high rate of cell division. In accordance with this type of distribution during interphase, it is not surprising to find that the values recorded for cells in the process of mitosis 40

Ha. of

nuclei 30

20

IO

DNA CONTtNT hridrary

Fig. l.-Interphase Experimental

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units)

nuclei.

523

DNA in rabbit kidney cells

show a single narrow peak at double the value of the lower limit of interphase, and corresponding to its upper limit. This would agree with the picture seen in a cell population of a single karyotype, although technical error could mask minor deviations should these be present. 40-

30-

No. of nuclei 20

1

IO-

20

40

60

I

80

ON1 CONTENT (arbiirary units) Fig. 2.-Mitotic

nuclei.

On comparison with control cells prepared immediately from fresh normal rabbit kidney and not cultured, it is seen that the whole range of values for RK13 cells is appreciably higher than that of the normal controls. This indicates a heteroploid complement of chromosomes.

DISCUSSION

Whilst these cells resemble those of other cell lines in being heteroploid, their distinguishing feature is the narrow restriction of the range of karyotypes. It would seem that the presence of a wide range of karyotypes, whilst being a common characteristic of converted cells, is not constant. The event of conversion from a cell strain to an established cell line is abrupt, and is accompanied by a change in karyotype from the diploid to the heteroploid state. It is assumed that variation in karyotype represents Experimental

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Sheila M. Sparshoff and E. S. Meek

underlying variation in genetic potentialities. The presence of a wide range of genotypes at the time of conversion of cells from those of a strain to those of a line would offer a basis for selection of the cell type most suited to in vitro conditions. In studies of the mitotic cycle calling for compartmentation of cells into Gl, S and G2 stages, this can be achieved by measurement of DNA content provided that the scatter around the modal value is negligible, as with diploid cells. Although RK13 cells are heteroploid, the restricted scatter resembles that of diploids and permits such staging to be carried out. The ready availability of cells of an established line offers an adv-antage over diploid cell strains when such investigations are to be pursued over a long period. The significance of heteroploidy in conversion to an established line requires some consideration, however. Whilst in vitro conditions cannot exactly duplicate the environment in uirlo, many types of cells can be maintained and will proliferate in vitro for periods ranging from a few kveeks to about a year. The presence of a diploid karyotype shows the continued close resemblance to the tissue of origin, and it may be postulated that variations can take place in the phenotype in the course of adaptation to an in vitro environment, subject, of course, to the underlying limitations of genotype. The changes in enzyme content which develop following explantation appear to be an example of this type of adaptation [3]. However, permanent adaptation appears to call for more profound changes than are possible within the framework of the original genotype. Here, the major changes associated with the onset of heteroploidy provide a mixed population of genotypes of which one may be much more successful in adaptation to the environment than either the original diploid stock or other genotypes. At this point the modal value of chromosome numbers characteristically alters from diploid to heteroploid. The continued presence of multiple karyotypes in established cell lines may indicate that the expressed characteristics of cells of the major karyotype are shared to a considerable extent by those with different chromosome numbers, so that a stable mixed population of metabolically similar cells results. Alternatively, the factors which gave rise to the major karyotypic disturbances at the onset of heteroploidy may still be operative SO that the production of variant cells derived from those of the modal karyotype continues indefinitely. The second possibility may be realized either through inherent instability of the chromosome complement or through environmental agents acting on it-intrinsic or extrinsic factors. Zamenhof [S] has shown that if bacteria are Experimental

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DNA in rabbit kidney cells

grown in the continued presence of a mutagenic agent, the mutation rate rises initially and then falls to the level found before the introduction of the mutagen, indicating a form of adaptation. On this evidence, it may be expected that, should some factor or factors capable of causing gross damage to the genome be present in the medium, continued growth in the same medium and conditions would lead by selection to the dominance of a cell type resistant to this action. Taking account of the long periods of time during which many established cell lines have been maintained, it would seem that environmental factors become of minor importance in relation to the continued emergence of variant karyotypes alongside the dominant type. Nevertheless, Saksela, Saxen and Penttinen [5] have shown that differences between individual sera added to the medium may lead to changes in chromosome numbers. In view of the number of sources from which established lines have been derived and the number of karyotypes which flourish in similar conditions and media, it is probable that the majority of cells with variant karyotypes are lineal descendents of those created at the time of conversion from the diploid to heteroploid state which were equally capable of continued proliferation. If this is so, then the distribution pattern of karyotypes seen in a cell line grown in replicate conditions over a long period will reflect closely the pattern present at the time of conversion. Differences between cell lines may thus represent not only differences of original source but also of the mechanism of heteroploidisation. It would be expected then that cell lines derived from a malignant tumour, such as HeLa, would show a greater diversity of karyotypes than those derived from normal cells, such as RK13, since these are the patterns seen in vivo. Nevertheless, this general persistence of the original karyotype distribution pattern may bc modified in two ways. At the time of conversion, it is probable that more cell types are present than are seen in later subcultures, since those capable of maximal growth rate will rapidly outstrip those with lower growth rates, these latter disappearing after a short period. In addition, some degree of inherent instability would seem to be involved as judged by the presence of abnormal mitoses. These form a potential source from which new karyotypes may be selected in the event of later changes in conditions of culture. From time to time it is to be expected, therefore, that there would appear cells with altered chromosome number; this would tend to modify the general pattern of karyotypic distribution to some degree over long periods. At present the establishment of a cell line depends either on spontaneous conversion Experimental

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Sheila M. Sparshotf and E. S. Meek of a cell strain occurring as a random event, or on the action of an oncogenic virus such as polyoma. From the foregoing it would seem that the RK13 line has resulted from a spontaneous event of mutation in a single cell from which all subsequent cells have been derived. In lines such as HeLa, derived from tissue which was malignant in uiuo, the agent causing the neoplastic change in the first place appears to have produced a number of different viable karpotypes which have persisted as a mixture of several different lines. There appear, then, to be t\vo distinct stages of adaptation to in vitro conditions. The first depends OII variation within the limits of the existing genotype; the second involves heteroploidisation and the establishment of a nen genotype or genotypes. SUMMARY

The distribution pattern of DNA values seen in an established line of rabbit kidney cells is discussed in relation to the variations of karyotype which occur during adaptation of cells to in vitro conditions. The authors wish to thank Mr C. Drown for technical assistance and to acknowledge generous financial support from the British Empire Cancer Campaign for Research. REFERENCES R. R. A., GURNER, B. IV., BEALE, A. J., CHRISTOFINIS, G. and PAGE, Z., Exptt Cell Res. 24, 604 (1961). 2. DEELEY, E. M., J. Sci. Instr. 32, 263 (1955). 3. GROPP, A. and HUPE, K., Virchotus Arch. pathol. Anat. 331, 641 (1958). 4. HAYFLICK, L., in Proc. Symp. of the Characteristics and Uses of Human Diploid Cell Strains, 1. COOMBS,

Opatija, 1963, p. 37. (Assoc. internat. des Sot. de Microbial.). 5. SAKSELA, E., SAXON, E. and PENTTINEN, K., Acta pathot. microbial. Stand. 51, 127 (1961). 6. ZAMENHOF, S., HELDENMUTH, L. H. and ZAMENHOF, P. J., Proc. Natl Acad. Sci. (Wash.) 55,

50 (1966).

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