Investigations into the mechanisms of carcinogen-induced nuclear enlargement in HeLa S3 cells in vitro

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Toxic. in Vitro Vol. 8, No. 5, pp. 1139--1150, 1994 Copyright © 1994 ElsevierScienceLid Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00

INVESTIGATIONS INTO THE MECHANISMS OF CARCINOGEN-INDUCED NUCLEAR ENLARGEMENT IN HeLa $3 CELLS IN VITRO C. WESTMORELAND*t, D. J. BENFORD*~, L. J. EALES§and P. GRASSO* *Robens Institute of Industrial and Environmental Health and Safety and §School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK (Received 8 November 1993; revisions received 16 February 1994)

A~traet--Carcinogen-induced nuclear enlargement has been reported both in vitro and in vivo, but the mechanism, and whether it is causally related to carcinogenesis, has not yet been established. This study was designed to investigate the role of increased DNA content, such as might occur in polyploidy, in induction of nuclear enlargement. The effects of two genotoxic carcinogens, N-methyl-N-nitrosourea and adriamycin, were compared with the effects induced by diethylstilboestrol, which is arguably a nongenotoxic carcinogen but is known to induce polyploidy. HeLa $3 cells were used as the model system for comparison with previous studies. N-methyl-N-nitrosourea and adriamycin both induced a concentration-related increase in nuclear size 24 to 72 hr after a 30 min pulse-treatment. This was accompanied by an increase in the proportions of cells in the G2 + M stage of the cell cycle, possibly due to a G2 block. There was some evidence of polyploidy with adriamycin but not with N-methyl-N-nitrosourea. The distributions of nuclear areas indicated that increases in ploidy contributed to, but did not totally account for, the nuclear enlargement. In contrast, diethylstilboestrol increased the range of nuclear areas and DNA content, to both less than and greater than that of control cells, but only after a prolonged exposure period of 48 hr. These data were consistent with diethylstilboestrol inducing spindle damage. These results demonstrate that carcinogen-induced nuclear enlargement is only partially explained by increased nuclear DNA content, and that certain classes of non-genotoxic carcinogen may produce a completely different pattern to that from genotoxic carcinogens.

INTRODUCTION Nuclear enlargement has often been observed as one of the earliest histological changes associated with chemically induced carcinogenesis in vivo, dating back to the work of Page (1938) who reported nuclear enlargement in the epidermis of mice following topical application of polycyclic aromatic hydrocarbon carcinogens. A large amount of literature now exists demonstrating nuclear enlargement after in vivo carcinogen treatment in many tissues including liver (e.g. Carriere, 1969; Clawson et al., 1992; Hendy and Grasso, 1977; Jackson, 1974), kidney (Hard and Butler, 1971), subcutaneous tissue (Hooson et al., 1971), intestine (Zedeck et al., 1970) and the nasal cavity (Fowlie et al., 1990) of experimental rodents. This phenomenon is seen almost exclusively in the organ selectively 'targeted' by the carcinogen for eventual tumour induction. tPresent address: In Vitro Toxicology Unit, C.P. Snow Building, University of Hertfordshire, Hatfield Campus, College Lane, Hatfield, Herts. ALl0 9AB, UK. :~To whom all correspondence should be addressed. Abbreviations: ADM = adriamycin hydrochloride; DES = diethylstilboestrol; DMEM = Dulbecco's minimal essential medium; MNU = N-methyl-N-nitrosourea; PBS = phosphate buffered saline; PI = propidium iodide.

The phenomenon of cr-cinogen-induced nuclear enlargement in mouse skin has been used to develop a short-term test for the evaluation of potential skin carcinogens (Ingram, 1979; Ingram, 1990; Ingrain and Grasso, 1977, 1983, 1985 and 1987). These studies have included tests with model carcinogens as well as with complex mixtures such as mineral oils containing polycyclic aromatic hydrocarbon carcinogens. The results have demonstrated that the measurement of nuclear enlargement is a sensitive and reliable method for the detection of potential carcinogenic activity in mouse skin. Although the correlation between the induction of nuclear enlargement and carcinogenicity is now well established in several organs, the nature of this enlargement is still far from clear in many cases. In the rodent liver, it has been demonstrated that a doubling of the nuclear volume is accompanied by a doubling of nuclear D N A content (Carriere, 1969; Clawson et al., 1992; Jackson, 1974). This relationship between nuclear size and altered cell ploidy has not been conclusively shown for all cases of carcinogen-induced nuclear enlargement, although an increase in size has often been taken as evidence for increased nuclear D N A . This is an important point to clarify, since both polyploidy and aneuploidy are known to be associated with the progression and

1139

1140

C. WESTMORELANDet al.

development of some tumour types (Brodsky and Uryvaeva, 1985; Dellarco et al., 1985). An in vitro model has previously been used to investigate further the nature of carcinogen-induced nuclear enlargement. An increased nuclear size was observed in HeLa cells after treatment with carcinogens in vitro. Grant and Grasso (1978) undertook a systematic investigation using a variety of compounds and found a good correlation between in vivo carcinogenicity and the extent of nuclear enlargement in HeLa cells in vitro. This was confirmed by further work of Agrelo (1978), Finch et al. (1980) and Agrelo and Amos (1981). In this publication we report results of an in vitro investigation of the relationship between nuclear enlargement and cellular DNA levels following exposure to carcinogens. HeLa $3 cells were used for comparison with previous data (Agrelo, 1978; Agrelo and Amos, 1981; Finch et al., 1980; Grant and Grasso, 1978), although it is recognized that transformed cells are not ideal for studies of aneuploidy and polyploidy, owing to the variability in chromosome number per cell. Three compounds with differing modes of action were selected for in-depth study: N-methyl-N-nitrosourea (MNU) and adriamycin hydrochloride (ADM) were chosen as examples of potent genotoxic agents, as well as diethylstilboestrol (DES), which is a human carcinogen of questionable genotoxic potential. MATERIALS AND METHODS Materials. HeLa $3 cells were kindly donated by Mrs D. Simpson of the School of Biological Sciences, University of Surrey, Guildford, UK and were routinely cultured as monolayers at 37°C. Cells were maintained in Dulbecco's minimal essential medium (DMEM) buffered with sodium bicarbonate. The medium was supplemented with 10% foetal calf serum, 100/zg kanamycin/ml and 10 mM L-glutamine. Culture media, sera, antibiotics and tissue culture flasks were obtained from Gibco (Paisley, UK). All chemicals were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated. Nuclear enlargement tests. The methodology used for nuclear enlargement assays was a modification of that used by Grant and Grasso (1978). Cultures of HeLa $3 cells were established on Thermanox cover-slips (Gibco) at a density of 1 × 105 cells per well in 12-well tissue culture plates, 24 hr before treatment with the appropriate chemicals. On the following day, the exponentially growing cells were exposed to the test chemical at the concentrations indicated in the Results. MNU was dissolved in phosphate buffered saline (PBS), ADM and DES were dissolved in dimethyl sulfoxide, which was added to PBS at a concentration of 1%. Control cultures were exposed to the solvent alone. Exposure was usually for 30 min with the exception of some experiments with DES where 3 and 48 hr incubations in DMEM were used.

Following exposure to the test chemical, cultures were washed twice with PBS and incubated for a further 24, 48 or 72 hr in fresh DMEM. Cells were then fixed using 3:1 (v/v) ethanol-glacial acetic acid and the cover-slips mounted onto glass slides. The nuclei were stained with gallocyanin/chrome alum (BDH, Poole, Dorset, UK) (Husain and Watts, 1983) after pretreatment in 1% potassium hydroxide in 80% alcohol to remove RNA (Ingram, 1990). The areas of at least 100 nuclei were measured from each of three coded replicate slides for each concentration/timepoint using a Quantimet Q920 image analyser (Cambridge Instruments, Cambridge, UK) by means of a microscope fitted with a x 40 objective and × 1.25 projection lens. Inter-phase nuclei only were measured and were selected on the basis that they were clearly separated from their neighbours. Statistical

treatment

o f data

on nuclear area.

Nuclear size data were generated in the form of a histogram, scoring frequency of areas in increments of 20 pm 2. Data from the three replicate slides were pooled for each treatment and the median and 99th percentile values calculated. The percentages of test cell nuclei that were larger than the 99th percentile of solvent controls were determined. Obviously, 1% of control nuclei are larger than the 99th percentile, and therefore an increase of 5% above the control 99th percentile was considered to have biological significance. In addition, the mean nuclear area from the three replicate slides was also calculated for each treatment and compared with the concurrent control mean, using Student's t-test. Flow cytometry. Cultures of HeLa $3 cells were established in 25 cm 2 flasks and 'pulse' treated with the test chemical in the same way as for the nuclear enlargement assays. 24, 48 and 72 hr following treatment, cells were rapidly fixed in cold 7:3 (v/v) ethanol-PBS and stored at 4°C for a minimum of 30 rain. DNA was stained for flow cytometry with the phenanthridinium dye propidium iodide (PI) using the methodology of Ormerod (1990). Fixed cells were washed with PBS and incubated for 30 min at 37°C with 100/~1 RNase (1 mg/ml; Boehringer Mannheim, Lewes, Sussex, UK) and 100 #1 PI (400/~g/ml). Flow cytometric analyses were made with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm argon-ion laser and FACScan research software. Red fluorescence was analysed from 10,000 cells per sample. In addition to fluorescence, the argon-ion laser was also used to generate forward and side scatter data for each cell, which are an indication of particle size and granularity, respectively. The relative percentages of nuclei in the G0/G~, S and G2 + M phases of the cell cycle were estimated for each control sample by electronic gating of the PI profiles. The relative percentages of cells in each phase for the treated samples at each time point were estimated using the electronic gating on the concurrent control. Nuclei located in the region

I141

C a r c i n o g e n - i n d u c e d n u c l e a r e n l a r g e m e n t in H e L a $3 cells Table 1. Induction of nuclear enlargement by N-methyI-N-nitrosourea (MNU) in HeLa $3 cells Nuclear area (/~m 2) Median

99th percentile

Percentage of cells > control 99th percentile

0 0.1 1.0 10.0

24

182.0 + 24.9 2110. + 26.2 206.3 __. 17.9 195.5 _ 12.1

181.7 204.2 199.8 193.8

364.9 438.3 430.6 431.2

1.0 3.6 2.7 3.1

0 0.1 1.0 10.0

48

193.5:[ 192.7 + 10.3 195.7 _+ 26.9 230.0:[

189.2 190.0 189.5 225.8

536.0 402.1 388.5 457.7

1.0 0.0 0.4 0.0

0 0.1 1.0 10.0

72

183.3 + 170.3 + 184.7 + 348.0 +

178.4 155.3 178.6 331.9

386.0 357.9 349.5 717.0

1.0 0.6 0.6 33.4t

0 50 500 5000

24

182.7 + 14.2 250.7 + 18.2"* 326.3 + 36.7*** 169.0 _+ 11.3

178.1 250.0 317.2 162.8

352.3 408.4 552.1 352.0

1.0 5.3t 36.It 1.0

0 50 500 5000

48

169.0 + 17.5 240.0 + 19.7"* 396.7 + 26.6*** 329.7 ___17.4"**

161.1 235.2 396.8 327.9

298.4 427.2 597.1 518.8

1.0 21.3~" 87.6t 64.0t

0 50 500 5000

72

236.0 369.7 472.3 438.0

219.2 346.8 461.0 436.3

470.3 728.3 907.9 717.4

1.0 18.7t 45.5t 34.1t

MNU (/~g/ml)

Time point (hr)

Mean + SD

23.0 25.7 12.2 41.4"*

+ 83.0 + 94.3 + 90.5** _+ I 1.1"*

tSignificance is attached to this value if more than 5% of cells have a nuclear area greater than the 99th percentile of the concurrent control. :[Only two replicate slides counted. Cells were pulse-treated with MNU for 30 min, then grown in control media until the time points shown. The areas of at least 100 nuclei on each of three slides were measured for each treatment group. Data are shown for two of three similar experiments with concurrent control values. Asterisks indicate significant differences from concurrent control (**P < 0.01; ***P < 0.001; Student's t-test).

Table 2. Induction of nuclear enlargement by adriamycin hydrochloride (ADM) in HeLa $3 cells Nuclear area (/zm2) Mean + SD

Median

99th percentile

Percentage of cells > control 99th percentile

0 2.5 5.0 10.0

24

161.3 + 22.6 223.7 +_ 28.7* 242.7 + 48.1" 351.7 + 33.0***

154.0 206.7 235.2 349.0

317.4 498.0 452.5 575.1

1.0 16. I t 15.5t 64.7t

0 2.5 5.0 10.0

48

182.7 +_ 31.0 305.0 + 64.9* 352.0 + 59.9** 404.0 + 47.9***

185.4 303.7 338.3 395.0

332.0 518.2 646.4 652.4

1.0 38.91" 52.3t 69.7t

0 2.5 5.0 10.0

72

169.0:[ 418.52 375.0 + 93.7 331.0 + 25.5

165.8 412.3 365.8 318.9

345.1 798.8 757.3 697.2

1.0 68.2t 54.9t 43.1t

0 0.1 1.0 10.0

24

182.0 + 15.4 221.7 __. 13.3"* 255.3 + 53.7* 305.7 ___59.1"*

175.2 222.2 242.3 302.9

310.1 433.4 455.8 522.9

1.0 9.3t 18.3t 47.2t

0 0.1 1.0 10.0

48

184.0 ___38.3 202.0 ___71.1 210.0 + 43.3 403.7 + 31.5"**

179.4 195.4 194.4 403.9

363.0 492.0 431.8 708.2

1.0 4.0 3.2 62.5t

0 0.1 1.0 10.0

72

150.7 + 18.0 152.3 + 18.7 148.0 + 22.9 332.7 _+ 29.4***

145.6 139.7 142.3 318.6

283.4 361.2 303.8 759.1

1.0 4.4 1.4 55.5t

ADM (/~g/ml)

Time point (hr)

*Significance is attached to this value if more than 5% of cells have a nuclear area greater than the 99th percentile of the concurrent control. :[Only two replicate slides counted. Cells were pulse-treated with ADM for 30 min then grown in control media until the time points shown. The areas of at least 100 nuclei on each of three slides were measured fur each treatment group. Data are shown for two of three similar experiments with concurrent control values. Asterisks indicate significant differences from concurrent control (*P < 0.05; **P0.01; ***P < 0.001; Students

t-test).

C. WESTMORELANDet al.

1142

beyond the G a u s s i a n distribution of G 2 ( > G2) were also measured as an estimation of the percentage o f polyploid cells. Three i n d e p e n d e n t replicate experiments were carried out for each c o m p o u n d . Commercially available cell cycle analysis packages could not be used, owing to the absence o f a G1/G0 peak in m a n y o f the samples. Chromosome preparation. In some experiments c h r o m o s o m e s were isolated from control a n d treated H e L a $3 cells using s t a n d a r d techniques. Cells were plated a n d treated as described above. 2 hr before harvesting, colcemid was added (final concentration 0 . 2 # g / m l ) to accumulate cells in metaphase. C h r o m o s o m e p r e p a r a t i o n s were stained using 10% G i e m s a (BDH), coded a n d analysed in r a n d o m fashion. A total of 200 metaphases were examined from each culture a n d the total n u m b e r of c h r o m o somes estimated for each metaphase. RESULTS

Nuclear enlargement M N U clearly induced nuclear e n l a r g e m e n t in H e L a $3 cells 24, 48 a n d 72 hr after 'pulse' treatm e n t for 3 0 r a i n with c o n c e n t r a t i o n s of 5-5000/~g M N U / m l , with the greatest increase in size seen after t r e a t m e n t with 500 # g / m l (Table 1). A preliminary investigation o f neutral red u p t a k e using the m e t h o d of B o r e n f r e u n d a n d Puerner (1985) d e m o n s t r a t e d t h a t M N U was cytotoxic at 5000 # g / m l , but not at 500/~g/ml (data not shown). T h u s the decrease in response at 5000/~g M N U / m l indicates t h a t the nuclear e n l a r g e m e n t is n o t associated with toxicity. The increases in nuclear area were greatest 72 hr after treatment. Lower c o n c e n t r a t i o n s o f 0 . 1 - 4 . 0 # g M N U / m l did n o t have any effect on the nuclear area o f H e L a $3 cells (data n o t shown). Figure 1 d e m o n strates the c o n c e n t r a t i o n a n d time relationship of the results from a representative experiment where H e L a $3 cells were treated with c o n c e n t r a t i o n s of 12.5, 25

a n d 50 # g M N U / m l . C o n t r o l H e L a $3 cultures gave good m e t a p h a s e p r e p a r a t i o n s with a m o d a l c h r o m o some n u m b e r o f 64. This c o m p a r e s well with the results of Sawada a n d Ishidate (1978), w h o quote a m o d a l c h r o m o s o m e n u m b e r for H e L a $3 cells of 6 2 ~ 3 . However, after 3 0 m i n treatments with all c o n c e n t r a t i o n s of M N U that induced nuclear enlargement, only metaphases containing extensively d a m a g e d c h r o m o s o m e s were present 24, 48 a n d 72 hr after treatment. C o n c e n t r a t i o n s of 10-500/~g M N U / m l led to an increase in cells in the metaphase stage of mitosis when c o m p a r e d with control cultures. This increase became most evident at the 72 hr sampling time. Nuclear enlargement was also observed in H e L a $3 cells 24, 48 a n d 72 hr after 'pulse' t r e a t m e n t with c o n c e n t r a t i o n s o f A D M ranging from 2.5 to 10/Lg/ml (Table 2). A t 1/~g A D M / m l , a small increase in nuclear area was observed 2 4 h r after treatment; however, this effect was reversed a n d no nuclear e n l a r g e m e n t was seen 48 a n d 72 hr after treatment. Figure 2 shows the results from a representative experiment where H e L a $3 cells were treated with c o n c e n t r a t i o n s o f 2.5, 5 a n d 10/~g A D M / m l . W h e n H e L a $3 cells were pulse treated for 30 min or 3 hr with c o n c e n t r a t i o n s of 0.3, 3 or 30/~g D E S / m l there was no consistent evidence of c o m p o u n d induced nuclear enlargement 24, 48 or 72 hr after t r e a t m e n t (data not shown). A small increase in the m e a n nuclear area was observed 72 hr after a 30 m i n t r e a t m e n t with 3 a n d 30/~g/ml. This increase was small a n d not reflected in the percentage of cells in these t r e a t m e n t groups with a nuclear area greater t h a n t h a t of the 99th percentile o f the c o n c u r r e n t control. Owing to the lack of any nuclear enlargem e n t o b t a i n e d using this m e t h o d o f 'pulse' treatment, alternative c o n c e n t r a t i o n s were used t h a t h a d previously been f o u n d to affect the c h r o m o s o m e n u m b e r o f H e L a $3 cells (Sawada a n d Ishidate, 1978). H e L a $3 cells were subsequently treated continuously with

Table 3. Induction of nuclear enlargement by diethylstilboestrol (DES) in HeLa $3 cells after 48 hr continuous exposure Nuclear area (/~m2) Percentage of DES cells > control (/tg/ml) Mean + SD Median 99th percentile 99th percentile 0 189.3 + 21.1 180.3 335.6 1.0 0.3 147.0_+ 19.1" 138.6 286.5 0.0 3.0 139.7 ___27.8* 131.9 323.9 0.7 30.0:~ 192.3 + 25.0 193.3 423.8 0.6 0 193.3 + 15.8 186.5 363.2 1.0 2.68§ 131.3 + 30.0 127.6 456.4 3.2 4.03§ 173.7 + 33.9 143.9 570.8 8.6% 5.37§ 209.0 _ 23.3 176.7 788.3 14.7% tSignificance is attached to this value if more than 5% of cells have a nuclear area greater than the 99th percentile of the concurrent control. :~Visiblycytotoxic concentration (only 262 nuclei could be measured). §Concentrations selected for comparison with literature data (Sawada and lshidate, 1978). Cells were continuously exposed to DES for 48 hr. The areas of at least 100 nuclei on each of three slides were measured for each treatment group. Data are shown for two of three similar experiments with concurrent control values. Asterisks indicate significant differences from concurrent controls (*P < 0.05; Student's t-test).

Carcinogen-induced nuclear enlargement in HeLa $3 cells

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Carcinogen-induced nuclear enlargement in HeLa $3 cells concentrations of 2.68, 4.03 and 5.37 #g DES/ml and the nuclear areas measured after 48 hr (Table 3). Using this treatment regimen, there was a dramatic effect on the number of large nuclei present, as indicated by a concentration-related increase in the 99th percentile value, with a concurrent increase in numbers of cells with small nuclei (Fig. 3). Overall, this resulted in a lack of effect on the mean nuclear area. These concentrations of DES were also used to confirm the finding of Sawada and Ishidate (1978), that DES affected HeLa $3 chromosome number. HeLa $3 cultures were treated with DES and chromosomes isolated after 48 hr. DES had a marked effect on the mean chromosome number per metaphase. The mean chromosome number 25 (a)

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( _ S D ) in control slides was 71.3_ 20.2. In slides treated with DES at 2.68, 4.03 and 5.37/~g/ml, the mean chromosome numbers ( + SD) were 83.2 + 29.2, 90.82 _ 25.3 and 98.7 + 28.5, respectively. Flow cytometry Cultures of HeLa cells were 'pulse' treated with concentrations of 0.1-5000/~g MNU/ml for 30 min. DNA per cell was analysed by PI fluorescence 24, 48 and 72 hr later. Figure 4 shows a set of histograms obtained after 48 hr in one of the three independent experiments conducted. Analysis of the data to give the percentage of cells in each stage of the cell cycle (Gt/G0, S, G2 + M or >G2) showed that, with increasing concentrations of MNU, there was a clear shift in the distribution of cells within the different stages of the cell cycle (Table 4). Significant increases were seen in the percentage of cells in G2 + M of the cell cycle, with a parallel decrease in the percentage of cells in stage G~/Go at each time point. At 72 hr a decrease in the percentage of cells in S phase was seen at concentrations of 10, 50 and 500 #g/mi. MNU did not induce a significant increase in the percentage of polyploid cells, although a small increase was seen in some experiments (see Fig. 4). A similar accumulation of cells in G2 + M was also seen 24, 48 and 72 hr after treatment of HeLa $3 cells with 2.5, 5 and 10/tg ADM/ml for 30 min. However, in this case there was also an increase in the percentage of ceils with a D N A content greater than that of cells in G2 + M, indicating the induction of a polyploid population (Table 5 and Fig. 5). When HeLa $3 cells were treated for 48 hr with concentrations of 2.68, 4.03 and 5.37/~g DES/ml, a dramatic effect was observed on the PI profiles of treated cells. Both the G~/Go and the G 2 -I- M peaks were greatly reduced, with a simultaneous increase in the percentage of cells with a > 6 2 DNA content and of cells with a DNA content of < G~ (i.e. DNA content less than 2n) (Table 6 and Fig. 6).

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IIIlllllllll

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Fig. 3. Nuclear area distributions of HeLa $3 cells treated with diethylstilboestrol (DES) at (a) 2.68, (b) 4.03 and (c) 5.37 #g DES/ml for 48 hr. Values represent the data from one of the experiments summarized in Table 3. The histogram shows the treated cell data; the concurrent control distribution is represented by the continuous line. T]V 81~-N

MNU, which is a classic genotoxic alkylating agent (IARC, 1987), induced nuclear enlargement in HeLa $3 cells over a wide range of concentrations and at sample times of up to 72 hr after a 30 min treatment. Analysis of the DNA content of these cells using flow cytometry indicated that this enlargement of cell nuclei is accompanied by a shift in the DNA content per cell from the control Gaussian distribution towards an increasing proportion of cells in G 2 + M of the cell cycle. The most likely expansion for these results is that MNU induces a G2 block in the cell cycle, a phenomenon that has been reported in other cell types after treatment with alkylating agents (Rao and Rao, 1976), rather than an induction of polypioidy. However, as the size of the nuclei of cells blocked in G2 greatly exceed the size distribution of control nuclei, other factors besides DNA content

C. WESTMORELAND et al.

1146 287

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Red fluorescence Fig. 4. FACScan propidium iodide fluorescence analysis of HeLa $3 cells 48 hr after 30 min pulsetreatment with N-methyl-N-nitrosourea (MNU): (a) control; (b) 10 pg MNU/ml; (c) 50#g MNU/ml; (d) 500/~g MNU/ml. Values represent the data from one of the experiments summarized in Table 4.

are likely to contribute to the enlargement. The relative decrease at the highest concentration was probably due to toxicity of MNU, as treatment at this concentration caused a decrease in neutral red uptake in a preliminary study (data not shown). ADM, which is also a potent genotoxin (IARC, 1987), also induced a concentration-dependent nuclear enlargement in HeLa $3 cells, confirming a similar finding by Lanks and Lehman (1990) in murine L929 cells. As with MNU, the increase in nuclear area was accompanied by an increase in the percentage of cells in G 2 + M. ADM and other DNA-intercalating agents are well documented to cause a 'G2 block' in many different systems (Kimler et al., 1978; Konopa, 1988). A second peak was also seen after treatment with ADM, showing the presence of a polyploid population. This may indicate that some of the cells blocked in G2 are released from the block to undergo further DNA replication without cell division. ADM has previously been reported to induce polyploidy in L929 cells (Lanks and Lehman, 1990) and murine erythroleukaemic cells (Zucker et al., 1991), possibly as a result of topoisomerase inhibition by ADM. A G2 block is a well-documented response seen in many cell types, which is often irreversible when caused by many antineoplastic agents, such as X-irradiation, neocarzinostatin and 1,3-bis-(2-

chloroethyl)-l-nitrosourea (Rao, 1980). This irreversible G2 block is usually accompanied by extensive chromosome damage, as observed in our studies with MNU. Other chemical agents can also arrest cells in G 2 in vitro, such as inhibitors of RNA/protein synthesis and amino acid analogues. Alterations in intracellular levels of cAMP can also affect the length of the G2 period; however, in most of these cases the G2 block is reversible. It has been hypothesized (Tobey, 1975) that the G2 phase of the cell cycle serves as a surveillance period to eliminate and modify cells with damaged DNA. This phase is then extended if the cells contain badly damaged DNA, as is the case after MNU treatment. This delay in the cell cycle can in some cases be indefinite. More recently, cell-cycle checkpoints have been implicated in the control of cell division (Murray, 1992). Such checkpoints detect the failure of cells to complete DNA replication, repair or spindle formation and arrest the progress of the cell cycle. Lesions in these feedback controls may be critical in the progression of carcinogenesis. It seems likely that cells blocked in G2 exhibit 'unbalanced growth' (Crissman et al., 1985) where RNA and protein synthesis continue in the absence of DNA synthesis. Such an unbalanced growth may be occurring after treatment of HeLa $3 cells with MNU and ADM, and may account for the increased nuclear area. Measurement of these nuclear

1147

Carcinogen-induced nuclear enlargement in HeLa $3 cells Table 4. Flow cytometry data of HeLa $3 cells pulse-treated with N-methyl-N-nitrosourea (MNU) Percentage of cells at each stage of cell cycle MNU (/ag/ml) 0 0.1 1.0 10 50 500 5000 0 0.1 1.0 10 50 500 5000 0 0.1 1.0 10 50 500 5000

Time point (hr) 24

48

72

Gt/Go

S

36.1 _+8.5 33.0_+4.3 32.0_+5.7 29.9_+7.2 18.4_+6.2' 7.6_+6.4** 32.3_+2.2 41.2 _+3.0 41.3_+3.3 37.2_+1.9" 8.0_+2.0*** 6.2_+2.8*** 3.0_+2.1"** 36.9_+7.6 41.9 _+3.4 44.5 _+3.4 40.1 +6.1 10.5_+3.1"** 4.0_+1.7"** 3.5 _+3.2*** 29.7 _+3.9*

20.0_+7.6 18.0_+6.4 19.3_+6.6 20.4_+6.4 13.9_+5.2 17.4_+5.2 16.2_+5.6 18.3 _+4.3 17.2_+3.0 16.9_+4.1 11.0_+5.9 15.4_+11.6 3.2_+0.1"* 16.5_+5.8 19.3 _+3.6 19.2 _+3.7 18.7-+3.3 8.6_+3.0** 7.6_+4.8** 4.6 _+ 1.5"** 14.9 _+5.2

G 2+ M 27.5_+0.5 28.8_+1.6 29.6_+2.7 28.8_+2.8 45.4_+2.4*** 53.6_+5.5*** 23.9_+5.3 22.9 _+5.0 23.5_+5.5 27.5_+5.5 57.4_+6.6*** 57.8_+13.8"* 66.7_+10.4"** 22.2_+2.9 19.6 _+3.8 18.4 4- 2.2 23.2+-4.4 53.4_+6.9*** 58.6_+9.8*** 69.2 _+8.3*** 23.8 _+4.5

>G 2 13.5_+3.9 14.1_+2.9 14.4_+3.9 15.7_+6.3 18.0_+6.4 15.1_+4.0 11.6_+3.2 13.3 _+3.6 13.8_+2.3 14.0_+1.8 18.5_+8.2 15.5_+1.8 22.5_+13.4 6.9_+2.3* 15.8 _+2.3 13.4 -+0.9 13.7-+2.6 20.5_+4.6 21.2_+4.3 15.4 _+ 1.0 6.6 _+ 1.4**

FSC 116.3_+3.2 113.7_+2.3 111.3_+0.6" 110.0_+2.6" 114.3_+5.0 110.3_+2.5" 91.3+20.4" 108.7 _+3.8 106.0_+4.6 108.3_+7.1 116.0_+4.4" 117.7_+5.5" 119.0_+1.7"* 78.0_+6.0*** 108.7 _+6.8 112.0 -+ 1.7 113.3-+2.1 19.7_+3.8"* 126.7_+10.5" 128.3 _+6.4** 75.7 _+6.4***

SSC 152.7_+ 16.8 156.3_+17.8 153.3_+14.6 152.7_+ 10.2 186.3_+11.7" 192.0_+24.5" 118.3_+23.7" 148.7 _+ 1.5 148.0_+7.8 162.7_+18.5 224.7_+26.3** 254.7_+33.3** 291.3_+5.9'** 107.7_+8.1"** 134.7 _+24.8 133.0 -+ 31.0 146.0-+25.9 273.0_+55.8** 293.0-+50.1"* 325.7 _+26.4** 109.3 _+ 11.7

FSC = forward scatter SSC = side scatter Cells were pulse-treated with MNU for 30 rain, then grown in control media until the time points shown. 10,000 cells per sample were analysed on the FACScan. DNA content was determined by the fluorescence of propidium iodide; forward scatter and side scatter indicate cell size and granularity. Values are means of three independent experiments _+SD. Asterisks indicate significant differences from concurrent control (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test).

p a r a m e t e r s in c o n j u n c t i o n w i t h n u c l e a r enlargem e n t tests m a y help in f u r t h e r u n d e r s t a n d i n g o f this problem. T h e c a r c i n o g e n i c s t a t u s o f D E S has recently been reviewed by M a r s e l o s a n d T o m a t i s (1992). D E S is a s s o c i a t e d w i t h the i n d u c t i o n o f vaginal a n d cervical clear-cell a d e n o c a r c i n o m a s in w o m e n e x p o s e d to the d r u g in utero. T h e g e n o t o x i c potential o f D E S , h o w e v e r , is still far f r o m resolved. C o n s i s t e n t l y negative results are o b t a i n e d w i t h D E S in m i c r o b i a l m u t a g e n i c i t y tests ( G l a t t et al., 1979; M c C a n n et al., 1975; Rfidiger et al., 1979) a n d the m a j o r i t y o f m a m m a l i a n cell m u t a t i o n a s s a y s ( B a r r e t t et al., 1983; B u e n a v e n t u r a et al., 1984; D e v o n et al., 1981; F r i e d r i c h a n d N a s s , 1983). O c c a s i o n a l positive test

results have been r e p o r t e d w h e n clastogenicity ( S a k a k i b a r a et al., 1991; S a t o et al., 1992), sister c h r o m a t i d e x c h a n g e ( A b e a n d Sasaki, 1977; D u l o u t a n d N a t a r a j a n , 1987; M e h n e r t et al., 1985; R/.idiger et al., 1979) a n d u n s c h e d u l e d D N A synthesis ( M a r t i n et al., 1978) h a v e been m e a s u r e d , a l t h o u g h negative results h a v e also been p u b l i s h e d f o r each o f these e n d p o i n t s . T h e results r e p o r t e d here c o n f i r m t h o s e o f S a w a d a a n d I s h i d a t e (1978), t h a t a 4 8 - h r t r e a t m e n t o f H e L a $3 cells w i t h D E S induces an increase in c h r o m o s o m e n u m b e r p e r cell. D E S h a s also been r e p o r t e d to induce b o t h p o l y p l o i d y ( B a n d u h n a n d O b e , 1985; D a n f o r d , 1985; D u l o u t a n d N a t a r a j a n , 1987; Ishidate, 1988; 1992; P r i s t o n a n d D e a n , 1985; S a t o et al., 1992) a n d a n e u p l o i d y ( B a n d u h n a n d O b e ,

Table 5. Flow cytometry data of HeLa $3 cells pulse-treated with adriamycin hydrochloride (ADM) Percentage of ceils at each stage of cell cycle ADM (/~g/ml)

Time point (hr)

0 2.5 5.0 10.0 0 2.5 5.0 10.0 0 2.5 5.0 10.0

24

48

72

G~/Go

S

G 2+ M

> G2

34.3+_2.6 3.6+_2.3*** 1.3+_0.6"** 0.5 _+0.1 *** 42.4_+4.4 2.7 +- 1.9"** 0.8_+0.3*** 0.6_+0.2*** 49.3_+3.8 1.2_+3.3"** 0.5 _+0.3*** 0.6_+0.1"**

25.7+2.4 6.6+_4.5*** 8.6_+10.0" 9.7 _+ 14.4 16.3_+3.5 2.7 _+0.6*** 1.8_+0.9"** 3.8_+5.4** 19.8_+5.0 5.2_+1.6"* 1.6 _+0.9*** 2.2_+ 1.7"*

24.2+3.3 67.4_+7.0*** 68.4_+8.8*** 70.3 _+ 15.6"* 21.5_+6.0 63.7 _+7.3*** 72.3_+7.1"** 73.8_+11.7"** 16.4_+5.0 50.9_+8.8** 74.6 _+7.2*** 77.3 _+7.5***

12.3+2.4 18.7_+3.6" 17.6_+0.8"* 16.4 _+2.5** 15.9_+2.0 26.8 _+8.9* 22.2_+5.9 18.9_+6.7 13.0_+4.2 32.1_+1.8"** 20.4 _+6.3 16.6_+ 1.3

FSC 85.3+_10.4 88.0+_I1.1 86.7_+5.7 87.7 _+7.3 93.0_+15.7 100.7 _+ 13.3 99.0_+13.0 97.3_+7.8 88.0_+ 11.1 96.3_+8.4 105.3 _+6.7* 100.7_+ 5.7

SSC 172.2-t-8.1 268.7___16.2"** 265.7_+13.0"** 264.3 +_ 12.9"** 169.0_+7.0 360.3 _+28.4*** 363.3_+5.5*** 333.7_+16.8"** 147.3_+21.4 393.3_+39.7*** 421.0 _+4.0*** 381.0_+ 18.3"**

FSC = forward matter SSC = side scatter Cells were pulse-treated with ADM for 30 min, then grown in control media until the time points shown. I0,000 cells per sample were analysed on the FACScan. DNA content was determined by the fluorescence of propidium iodide; forward scatter and side scatter indicate cell size and granularity. Values are means of three independent experiments __.SD. Asterisks indicate significant differences from concurrent control (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test).

1148 287

C. WESTMORELANDet al.

(a)

(b)

(c)

II (d)

m

0 287 E z

0

~

~

0

1023 0

1023

Red fluorescence

Fig. 5. FACScan propidium iodide fluorescence analysis of HeLa $3 cells 48 hr after 30 min pulse-treatment with adriamycin hydrochloride (ADM): (a) control; (b) 2.5 #g ADM/ml; (c) 5.0#g ADM/ml; (d) 10/~g ADM/ml. Values represent the data from one of the experiments summarized in Table 5. 287 (a)

(b)

(c)

(d)

m

o 287 es

Z

0

0

760 0 Red fluorescence

Fig. 6. FACScan propidium iodide fluorescence analysis of HeLa $3 cells after 48 hr continuous treatment with diethylstilboestrol (DES): (a) control; (b) 2.7/~g DES/ml; (c) 4.0 #g DES/ml; (d) 5.4 #g DES/ml. Values represent the data from one of the experiments summarized in Table 6.

760

Carcinogen-induced nuclear enlargement in HeLa $3 cells

1149

Table 6. Flow cytometry data of HeLa $3 cells treated with diethylstilboestrol (DES) for 48 hr Percentage of cells at each stage of cell cycle DES (#g/ml) 0 2.68 4.03 5.37


G I/GO S G 2+ M >G 2 FSC SSC 42.5_+3.2 18A + 1.4 28.3+3.5 9.7_+3.6 112.3_+0.6 150.0_+1.7 15.9 _+1.7"** 13.6 _+ 1.6"* 13.8 + 0.7*** 30.1 __.4.7** 99.7 __.4.9** 142.0 _ 3.0** 8,9 + 3.6*** 12.1 -+ 3.6* 19.3 -+ 6.7* 39.2 -+ 10.5"* 100.0 -+6.0** 168.3 _+19.9 8A _+2.2*** 10.4 + 2.4** 17.4 + 3.3** 43.7 + 8.4*** 95.3 -+ 5.8*** 157.7 _+ 1.7 FSC = forward scatter SSC = side scatter Cells were exposed to DES continuously for 48 hr. 10,000 cells per sample were analysed on the FACScan. DNA content was determined by the fluorescence of propidium iodide; forward scatter and side scatter indicate cell size and granularity. Values are means of three independent experiments + SD. Asterisks indicate significant differences from concurrent control (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test). 1985; D a n f o r d , 1985; D u l o u t a n d N a t a r a j a n , 1987; Sato et al., 1992; Tsutsui et al., 1983) in a variety of cell types in vitro. It has been suggested (Hartley-Asp et al., 1985; S a k a k i b a r a et al., 1991; S h a r p a n d Parry, 1985) t h a t the effect of D E S o n cellular c h r o m o s o m e c o m p l e m e n t s m a y be a result o f spindle damage, as D E S is k n o w n to inhibit the in vitro polymerization of m i c r o t u b u l e proteins. This effect of DES o n c h r o m o s o m e n u m b e r was reflected in the results o f the analysis o f nuclear size a n d D N A content of treated cells. A n increase in b o t h large a n d small H e L a $3 nuclei was observed after t r e a t m e n t with DES, which was a c c o m p a n i e d by a n increase in cells with a D N A c o n t e n t o f < 2n a n d > 4n as m e a s u r e d by flow cytometry. F u r t h e r investigations o n the effects o f D E S in a finite cell line with stable c h r o m o somal n u m b e r are required to clarify the role o f aneuploidy in these observations. In conclusion, M N U , a potent genotoxic carcinogen, induced a concentration-related nuclear enlargem e n t in H e L a $3 cells pulse treated for 30 min. This was a c c o m p a n i e d by a n increase in the p r o p o r t i o n s o f cells in the G 2 phase of the cell cycle, b u t not by polyploidy, suggesting t h a t other factors t h a n D N A contribute to the nuclear enlargement. A similar picture was o b t a i n e d with A D M , a l t h o u g h in this instance there was evidence of induction of polyploidy. DES, a chemical of equivocal mutagenic properties, required a m u c h longer exposure (48 hr) to produce a n effect o n H e L a $3 cells. The distrib u t i o n of nuclei o f DES-treated cells showed increases in b o t h large a n d small nuclei, so t h a t the m e a n value was n o t altered. This effect is likely to reflect spind!e damage.

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