Induction of polyploidy by concentrated thymidine

Experimental Cell Research 68 (1971) 442-448

INDUCTION

OF POLYPLOIDY

BY CONCENTRATED

THYMIDINE

C. G. POTTER Cytogenetics

Unit, Nuffield

Wing, School of Medicine, Liverpool, UK

University

of Liverpool,

SUMMARY Normal human skin fibroblast cells exposed for more than about 17 h to thymidine at concentrations above 1 mM showed an increase in polyploid index over control levels. This increase, in which cells with even polyploid numbers predominated, varied up to a maximum of 36 %. The thymidine-treated cultures also show considerable chromosome damage in both diploid and polyploid cells.

In the last decade a number of papers have appeared which have been concerned with the effects of millimolar concentrations of thymidine (TdR) on cultured cells. The most important effect is that of inhibition of DNA synthesis [19] due to the large amounts of thymidine triphosphate end product, which strongly inhibits the cytosine monophosphate to deoxycytosine monophosphate pathway [24, 251. This phenomenon was used originally by Xeros [30] to produce partial synchrony in Chang appendix cells. Since then work on the optimisation of synchrony by this method has been performed on a variety of cell lines, including a heteroploid human kidney line [4, 8, 91, HeLa cells [6, 17, 18, 23, 28, 291 L5178 mouse lymphoma [5], CMP adenoma [15], hamster ovary cells [21] and phytohaemagglutinin-transformed human leucocytes [27]. Toxic effects were noted in synchronised L5178 cells [5] while plating efficiency was reduced in HeLa cells after 24 h exposure to concentrated thymidine [16], although synExptl Cell Res 68

chronous growth could still occur after exposures of up to 90 h [17]. Chromosome abnormalities have been studied in aneuploid Chinese hamster cells by Yang, Hahn & Bagshaw [31] at the first mitotic peak after release from a thymidine block. The proportion of cells affected by millimolar thymidine varied between zero and 51.3 “/b but in any one experiment the percentage of affected cells increased with the increase in concentration. It may be relevant that Mg2+ DNA-ase activity is high in HeLa cells exposed to concentrated TdR as this enzyme is active against denatured but not native DNA [I 7, 18, 291. In the earlier work there was only occasional reference to morphological defects although Bootsma, Budke & Vos [4] noted some scattering of chromosomes during metaphase and Firket & Mahieu [6] found some chromosomal and mitotic anomalies in HeLa cells when a double block was used; they also found that the time of metaphase was extended. This latter phenomenon has

Polyploidy induction by thymidine been studied by Izutsu & Biesele [14] in HeLa cells exposed to 2-4 mM thymidine, when metaDhase was found bv time lanse photography to be significantly increased, while other phases of mitosis were unaffected. This result-was earlier shown by Barr [2] using conventional differential mitotic index counts, also using HeL. cells. He suggested that metaphase delay could account for the increased mitotic index found by Greulich et al. [IO, 1 I] in the in vivo studies on the effect of thymidine, at concentrations of the order of 0.3 ,ug/g body weight, on proliferating mouse duodenum. A similar metaphase prolongation has been shown in Happlopappus gracilis root tip material using a medium containing 2.9 x 10PG M thymidine [I]. This present work is a study of the increase in polyploidy and occurrence of chromosome abnormalities in normal human skin fibroblasts after treatment with concentrated thymidine. These phenomena were first noted incidentally during relatively unsuccessful attempts at synchronising this type of cell culture. i

I

MATERIALS

AND

I

METHODS

Tissue culture Five different normal human diploid skin cultures were used (lines A-E). The cells were grown in 5 cm Carrel flasks by a modification of Harnden’s method [II]. The medium which was used comprised 70% TC 199, 20 96 human AB- ve serum and IO 96 chick embryo extract in all exoeriments exceut IIB where 20 :b foetal calf serum (Flow Labs) and’80 “:, TC I99 were used.

Chromosome preparation and counting Cells were pretreated with Colcemid (0.001 7;) for 3-4 h and trypsinised off the glass. The suspension was made hypotonic by the addition of distilled water (3: I) and after swelling for 5 min the cells were spun down, fixed in ethanol/acetic acid (3: I), and slides made by an air drying technique. To prevent bias against poorly spread cells all the metaphases encountered in systematic scanning were scored for polyploidy using low power magnification ( I 150). For determination of polyploid level a rough count at 675 was usually sufficient to place polyploids in ?!I

711X14

443

their correct class but aneuploid cells with chromosome numbers widely different from any particular uloidv level were ulaced in the next higher nloidv . , class,- as these cells were regarded as either being burst during preparation, or iesulting from chromesome loss in culture. Cells with up to three chromosomes above an exact ploidy level were classed at that level

Microspectrophotometry Cells on coverslips in Leighton tubes were given I &i/ml 3H-TdR for 4 h before fixation in 95 Y:, ethanol. The cells were stained by the Feulgen reaction before autoradiography using AR 10 stripping film followed by exposure for 6 days and processing. Only unlabelled cells were read (using a GN 2 Barr and Stroud microdensitometer) as, apart from inactive cells, these comprised only G 1 cells, since during the 4 h exposure to 3H-TdR most of the G2 cells divide and enter the G 1 population [22].

General plan of experiments Thymidine (British Drug Houses) was made up IO concentration in Hanks solution and the appropriate amount added to the cultures. After exposure it was removed by rinsing three times with Hanks before replenishing the medium and regassing. After different experimental treatments with thymidine different levels of synchrony were expected within any experiment. The cultures were therefore left for several days after treatment for any synchrony to decay to a negligible level. By this time the cultures were generally overgrown so subculturing was necessary before harvesting for chromosomes.

RESULTS

1. Effect of thymidine concentration on polyploid level Two experiments were performed. The first on Line A covered a wide range from 10 mM down to 10~~ mM plus untreated controls. Two cultures were provided for each treatment consisting of 6.2 x 10” cells seeded into 5 cm Carrel flasks. After 30 h exposure and a week of culture, followed by subculturing, there was a total of four cultures for each treatment. Samples of 200 cells from each culture were examined for polyploids giving 800 cells for each point of the line as shown in fig. 1 A. Only cells treated with 10 mM or I mM showed any perceptible increase in polyploid index over the controls. Exptl Cell Res 68

444

C. G. Potter

zo-

lo-

lo-

t---,+ ,i

+I 0.001

m-J 0.01

0.1

+ Control* I 1.0

1 10.0

Fig. 1. Abscissa: TdR concentration (mM); ordinute: ~4,polyploids. Variation of polyploid index of human skin fibroblasts exposed to different concentrations of thymidine for 30 h. (kS.E. of the proportion).

5-

0I,

0

I

10

I

20

I

30

Filr. 2. Abscissn: exoosure time (hours): ordinate: Y, polyploids. Variation of oolvoloid index of human skin fibroblasts exposed ’ to- ‘5 mM thymidine for different times. (+S.E. of the proportion).

In the second experiment, a narrower range of concentration was used on line 9. 2.8 x: IO5 cells were seeded into each Carrel flask and they were again exposed for 30 h to the giving I 600 cells for each point of the line various concentrations of thymidine. This time a total pooled sample number of 600 cells shown in fig. 2. A poorly defined threshold per point was used as shown in fig. 1 B. In can be seen at about 17-20 h exposure to the this experiment a definite threshold can be 5 mM thymidine. seen whereby a concentration of thymidine of 2 mM or greater produces an increase in (B) A similar experiment on line E was performed except that the cells were harvested the polyploid index. so a lower cell In these experiments the concentrations of for microspectrophotometry density was used in setting up (6.5 x IO3 cells/ thymidine that produced increases of polyploid index are similar to those required in ml). Samples of 200 cells were examined for each histogram in fig. 3. Again the main other lines to inhibit DNA synthesis, apart increase in polyploidy occurs after about 16 h from one unusually sensitive strain of CCRFexposure to 5 mM thymidine. Also evident CEM human lymphoblast cells [7, 261. are many amodal cells, presumably aneuploid, II. Effect of different exposure times to especially at the higher exposure times. thymidine

on polyploid

lecel

(A) Cultures of line B were set up with 2 x lo5 cells per Carrel flask. After 24 h thymidine was added to a final concentration of 5 mM to all the cultures apart from two controls which were rinsed and the medium renewed. At various times after the addition of the thymidine two samples were rinsed and allowed to continue growth until subculture and chromosome preparation. Four hundred metaphases were examined from each culture Exptl Cell Res 68

I I I. Types of polyploid trated thymidine

produced by concen-

Two experiments were performed using different cell lines. In the first experiment the cells (line D) were exposed to 5 mM thymidine for 50 h while in the second experiment (line 9) the same concentration was used but only for 30 h. The same general plan was used as previously described. Six hundred metaphases were examined from treated and

Polyploidy

induction

by thymidine

445

Fig. 3. Ahscis.sa: DNA level in arbitrary units, on a log, scale; ordinate: oo polyploids.

Microspectrophotometric times.

data on 3H-TdR-labelled samples of cells exposed to 5 mM thymidine for different

control cultures for the first experiment and 1 600 from each for the second experiment. The results are shown in fig. 4a, 6. There is a much greater increase in polyploidy in line D (35.9 “A), which had the longer exposure time, compared with line B (8.9%). In both, however, the even ploidy levels predominate as would be expected if doubling of the chromosome number had occurred. If this were so, then the higher ploidy levels could have been derived from polyploids already present, as in the control cultures. The considerable number of cells in the 3N class are probably aneuploid or broken cells as 10 karyotypes, made after re-examination of these cells, showed no exact triploids or cells based on the triploid complement. In contrast a sample of 10 karyotypes from

the 4N class were all recognisably tetraploid, despite the presence of aberrations and slight aneuploidy. 1V. Chromosome aberrations concentrated thymidine

induced by

In the previous experiments many aberrations were seen in the thymidine-treated cultures. Therefore a more detailed examination was made of a sample of well spread metaphases from the second experiment in 111and the results shown in table 1. Chromatid breaks, isochromatid breaks and exchanges were noted as well as rings, dicentrics and fragments. In this preliminary investigation karyotypes and precise details of chromosoma1 rearrangements were not investigated as relevant information of this type should come Exptl Cell Res 68

446

C. G. Potter shown in fig. 5 6. Chromatid exchanges are shown in fig. 5c, a dicentric in fig. 5n’ and a complex exchange in fig. 5e. There was no consistent feature of diplochromosomes or chromosome pulverisation, the latter being noted in l-2 “6 of thymidine-treated Chinese hamster cells [31].

20-

15-

lo-

5-

o-

--

3

4

DISCUSSION 10 a

6f 4 2 0i 3

4

5

6

345676

16

Fig. 4. Abscissa: ploidy level, N; ordinate: % polyploids. (a) (left) control (total 1.8 % m=11 cells); (right) treated with mM TdR (total 37.7 % ~ 226 cells); (b) (left) control (total 5.0 % ~ 80 cells); (right) treated with mM TdR (total 13.9 % ~~223 cells). Variation of ploidy levels in human skin fibroblasts after exposure to 5 mM thymidine as compared with untreated controls. (a) 50 hours exposure to TdR on strain D. (b) 30 hours exposure to TdR on strain B.

from cells in their first division after treatment. An extreme example of the types of abnormalities seen is shown in fig. 5a while a good example of a ring chromosome is

Polyploidy can arise as a result of primary or secondary effects. As detailed in the introduction, thymidine can prolong metaphase, perhaps by acting as a spindle poison such as colchicine and thereby inducing polyploidy in a similar manner. The long exposure times and the high concentration threshold required are however more in accordance with the polyploidy being associated with the thymidine block. Another primary effect would be if a further S period occurred in some cells as an adjustment to the inappropriate cytoplasm to nuclear ratio resulting from a period of unbalanced growth of cells in concentrated TdR. As a secondary effect it is known that radiation induced aberrations are followed by polyploidy in leucocyte cultures [3, 13, 201, possibly due to mechanical difficulties

Table I. Proportions of aberrant cells after thymidine treatment Polyploid 2N CLASS’ 3N CLASSa 4N CLASSa 5N CLASSa 8N CLASSa Total Control

Z/l00 (2.0 46)

l/2

Tdrtreated

32/167 (19.2 %)

419

112

16/3l

a Ploidy classes include aneuploids down to the next euploid number Exptl Cell Res 68

-

3/lO (30.0 Yb) Equiv. to 3/20 diploid sets (I 5.0 X) 21/41 (51.2 ‘xv:,) Equiv. to 21179.5 diploid sets (26.4 ?A)

l/l

3.

Total 5/l IO (4.5 9”)

53/208 (25.5 ?o)

Polyploidy induction by thymidine

447

Fig. 5. Examples of chromosome damage by concentrated thymidine (5 mM for 30 h). (n) Numerous dicentrics and chromosome breaks in a highly aneuploid cell. (b) a ring chromosome and chromosome breaks; (c) detail of a cell showing chromatid exchanges; Cd) a dicentric; (E) detail of a complex exchange.

448

C. G. Potter

of aberrant cells at anaphase, although not all of the endoreduplicated cells after such treatment show chromatid aberrations [20]. The increase in polyploidy after thymidine treatment could be explained in a similar way especially as the data (table I) does show some increase in aberration frequency per chromosome set in the polyploids compared with the diploid cells. It is also possible however that polyploids with their redundancy of chromosomal material may be more likely to survive than diploids, despite proportionally more chromosome damage. Such a mechanism could also explain some of the increase in polyploidy by selective diploid death. However, very many diploids must be killed to produce even a moderate increase in polyploid index and no signs of such high death rates were noticed in these experiments. Less noticeable selection over several cell cycles could however occur during the time taken by this type of experiment, so resulting in a high selection rate overall. Either theory is compatible with the exposure time and concentration effects, as aberrations or death rate could be a function of thymidine block. It may also be significant that thymidine induced synchrony of normal human diploid material does not appear to have been reported in the literature despite the relative ease and universal cultivation of such cells. My own (unpublished) attempts show a poor proportion of synchronised cells after release from thymidine block and this may be partly explained by the chromosome damage and polyploid production demonstrated by this work. This work was financed by a grant from the United Sheffield Hospitals Endowment Fund and by the North West Cancer Research Fund, UK.

Exptl

Cell Res 68

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