Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids.

INT . J . RADIAT . BIOL .,

1991, VOL . 60, NO . 3, 453-466

Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids P . L . OLIVEt , R . E . DURANDt, J . P . BANATHt and H . H . EVANS§

Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 01/06/15 For personal use only.

t B .C . Cancer Research Centre, Vancouver, BC, Canada § Case Western Reserve University, Cleveland, Ohio, USA (Received 31 August 1990 ; revision received 5 February 1991 ; accepted 2 April 1991)

Chinese hamster V79 cells grown in suspension culture as spheroids are more resistant than monolayers to killing and mutation by ionizing radiation . A change in DNA conformation appears to accompany this increase in radiation resistance . We were therefore interested in whether the activity of topoisomerase II, a nuclear enzyme involved in DNA conformational changes and possibly in DNA repair, might differ in monolayers and small spheroids . One-day-old spheroid cells were more resistant than monolayer cells to the toxic effects of etoposide, a topoisomerase II inhibitor . Fewer strand breaks were induced by etoposide in spheroid DNA than monolayer DNA, as measured by the DNA precipitation and alkali unwinding assays, although identical amounts of damage were produced in monolayers and spheroids by the topoisomerase I inhibitor camptothecin, and the cell cycle specific agent, 5-fluorouracil . There was no evidence of a subpopulation of spheroid cells which were more resistant to etoposide, and no change in the rate of incorporation or DNA chain elongation in spheroids compared to monolayers. Topoisomerase II activity in 1-day-old spheroids, measured by decatenation of trypanosome kinetoplast DNA, was reduced to 68% of the monolayer value ; in 3-4-day-old spheroids the level was 32 . 5% . These results indicate that topoisomerase II activity and sensitivity to a topoisomerase II inhibitor are reduced in 1-day-old spheroid cells . We suggest that the decrease in the activity of this enzyme may be linked to the change in DNA conformation in spheroids and the decrease in their radiation sensitivity.

1.

Introduction

Chinese hamster V79 cells grown for 24 h in suspension culture initially aggregate and then divide to form clusters of 20-50 cells . Cells from spheroids are more resistant to killing and mutation by ionizing radiation than are cells grown as monolayers (Durand and Sutherland 1972, 1973, Olive and Durand 1985) . This observation has been called the `contact effect' (Durand and Sutherland 1972, 1973) . The explanation for the increased radiation resistance of spheroid cells is not known, although there is reason to suspect that changes in DNA `packaging' or nuclear morphology may be involved (Olive et al . 1986, Gordon et al . 1990) . One-day-old spheroids show unusual DNA denaturation kinetics when lysed in 1 mI NaCl and 0 . 03 mI NaOH . Unlike DNA from irradiated monolayer cells which unwinds exponentially, irradiated spheroid DNA stops unwinding after 5-10 min :To whom all correspondence should be addressed at Medical Biophysics Unit, BC Cancer Research Unit, 601 W 10th Avenue, Vancouver, BC, Canada V5Z 1L3 . 0020-7616/91 $3 .00 © 1991 Taylor & Francis Ltd

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(Olive and Durand 1985, Olive et al. 1986) . On the basis of this observation it was suggested that there may be `constraints' to DNA unwinding present in DNA from spheroid cells, spaced at regular intervals about 50µm apart (Olive et al . 1986) . Since the development and loss of constraints coincided with the development and loss of the `contact effect' in terms of cell survival (Olive and Durand 1985, Olive 1989), we proposed that these constraints may somehow be involved in the contact effect . The change in DNA conformation which is associated with growth of cells in suspension could also be reflected in, or caused by, a change in the activity of enzymes that control and modify the topological states of DNA . Topoisomerase II is an enzyme catalysing concerted DNA strand breaking and rejoining reactions which are an essential part of DNA replication (Liu et al. 1980, Wang 1982) . The activity of this enzyme is associated only with actively proliferating cells (Sullivan et al . 1986, Hsiang et al . 1988) . Evidence suggests that topoisomerase II mediates double-stranded DNA cleavage by making two coordinated single-stranded breaks in the nucleic acid backbone (Zechiedrich et al. 1988) . These enzyme molecules are believed to be a part of the scaffold proteins in mitotic chromosomes and are located at the base of the DNA loops (Earnshaw et al . 1985), reminiscent of the distribution of constraints to DNA unwinding in spheroids . There is also growing evidence indicating that topoisomerase II may play a role in DNA repair following radiation damage . L5178T-S (LY-S) lymphoma cells are more sensitive to killing by ionizing radiation than are L5178Y-R cells, and LY-S cells are also more sensitive to topoisomerase II inhibitors (i .e . m-AMSA, etoposide) which stabilize the cleavable complex between DNA and the enzyme (Evans et al . 1989) . The LY-S cells also exhibited a higher level of DNA doublestrand breaks following m-AMSA treatment than the LY-R cells (Evans et al . 1989) . A definite correlation between the sensitivity to ionizing radiation and to topoisomerase II inhibitors was demonstrated by sublines of strain LY-S which were selected for increased resistance to ionizing radiation and which also showed increased resistance to topoisomerase II inhibitors (Evans et al. 1989) . Ataxia telangiectasia fibroblasts which are 3-4 times more sensitive to killing by ionizing radiation than normal diploid fibroblasts are also more sensitive to topoisomerase I I inhibitors (Henner and Blazka 1986, Smith et al . 1986) and show higher levels of topoisomerase II (Davies et al . 1989) . Finally, some Chinese hamster mutant cell lines hypersensitive to ionizing radiation have been found to be more sensitive to topoisomerase II inhibitors (Robson et al. 1987, Jeggo et al . 1989) . Based on these observations, we examined the hypothesis that topoisomerase II activity is altered in V79 spheroids compared to monolayers, and that the change in this enzyme might therefore be related to DNA conformation and radiation sensitivity. Topoisomerase II activity was measured in V79 monolayers and spheroids directly, using the trypanosome kinetoplast DNA decatenation reaction, or indirectly by measuring DNA strand breakage and cell killing by etoposide . 2.

Materials and methods

2.1 . Cell culture conditions Chinese hamster V79 lung fibroblasts were maintained in exponential growth by subculturing twice weekly in minimal essential medium containing 10% fetal bovine serum from Gibco, Grand Island, NY . Single cells (2 x 10 5 cells/ml) were exposed to etoposide (Bristol Laboratories) for 1 h at 37°C in a humidified CO 2

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incubator. Following incubation, cells were again trypsinized and cell survival was measured using a standard clonogenicity assay . Colonies were stained and counted 1 week later . Samples of cells were also routinely examined by flow cytometry to ensure that the distribution of cells in the growth cycle was the same for the monolayers and 1-day-old spheroid cells .


2 .2 . DNA precipitation assay The DNA precipitation assay was used to detect DNA single-strand breaks (ssb) (Olive 1988) . This method measures the fraction of DNA which precipitates when sodium dodecyl sulphate (SDS) is used to lyse the cells and potassium chloride is added to cause precipitation of the detergent, cellular protein and large molecular weight DNA . Monolayers were incubated for 24h with 0 . 7 kBq/ml [ 14C]thymidine . Cells were then trypsinized and placed into monolayer culture or into spinner culture flasks for growth as spheroids . After 24 h, monolayers and 1-day-old spheroids were trypsinzed, exposed to etoposide for 1 h, then resuspended in complete medium at a density of 10 6 cells/ml . Cell suspension (100 µl) was then placed in tubes (in triplicate) and 0 . 5 ml of alkaline lysing solution (2% SDS, 10 mm Tris, 10 mm EDTA and 0 . 05 M NaOH) was added . After 1 min, 0 . 5 ml 1-2m KCl was gently added and a white precipitate immediately formed . Tubes were carefully placed in a water bath and heated for 10 min at 65°C . DNA and protein were then precipitated by placing tubes on ice for 5 min . Tubes were centrifuged for 5 min at 1800g . The supernatant fluids were poured into scintillation vials and neutralized with 1 ml 0 . 03 M HC1 . The pellet was redissolved in 2 ml hot water, and added to a second scintillation vial . Hydrofluor liquid scintillation fluid was added, radioactivity counted using a LKB 1217 liquid scintillation counter, and the percentage of DNA precipitated was calculated as the dpm in the pellet divided by the total dpm in the pellet plus supernatant multiplied by 100 .

2 .3 . Comet assay For measurement of DNA damage to individual cells we used the microelectrophoresis method first described by Ostling and Johanson (1984) and recently modified to improve sensitivity for detection by video image analysis (Olive et al . 1990a,b) . Monolayers and spheroids were trypsinized, exposed for 1 h to 2 µg/ml etoposide, and resuspended as a single cell suspension in phosphate buffer . Cells (10 4) in 0 . 5 ml were mixed with 1 . 5 ml melted 1 % agarose (Sigma 6013) then rapidly spread on a microscope slide . Slides were lysed for 1 h in the dark in a solution containing 0 . 03 M NaOH and I M NaCl . After lysis, slides were briefly rinsed in distilled water and subjected to electrophoresis at 4 V/cm for 12 min in a Tris/EDTA/acetate buffer, pH 8 . 3 . Slides were stained for 10 min in 2 . 5µg/ml propidium iodide and individual cells were analysed under 546 nm light excitation using a fluorescence image processing system . Each cell was seen as a `comet' with a brightly fluorescent head and a tail whose length and intensity of fluorescence was proportional to the amount of damage induced by etoposide (Olive et al . 1990b) . The tail moment, proportional to the fraction of DNA in the tail of the `comet' and the length of the tail, was calculated for 400 individual cells, chosen at random from the centre of the gel . Identification and analysis required only a few seconds per comet; data reduction and background correction have previously been described (Olive et al . 1990a) .

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2.4.

Alkali unwinding assay 14C-labelled monolayer and spheroid cells (prepared as described for the DNA precipitation assay) were incubated with etoposide then resuspended at a density of 2 x 10 5 cells/ml . Cell suspensions (50µl) were placed in tubes, and 1 ml of freshly prepared lysing solution was added (0 . 03 M NaOH, I M NaCl) . After I min to 2 h of lysis in the dark, on ice, samples were neturalized with 1 ml 0 . 03 M NaH 2 PO4 and immediately sonicated . Single- and double-stranded DNA fragments were separated using hydroxyapatite chromatography (Ahnstrom and Erixon 1974), and samples were counted using a LKB 1217 liquid scintillation counter . The percentage of double-stranded DNA (%DS-DNA) was calculated as the dpm in the double-stranded fraction divided by the dpm in the single plus double-stranded fractions multiplied by 100 .

2.5.

Incorporation and elongation Incorporation of radiolabelled thymidine into DNA was measured using monolayers and spheroids which had been labelled for 24h with 0 . 7 kBq/ml [ 14 CJthymidine . Single cells were prepared and incubated with 37 kBq/ml [ 3 H]thymidine . Samples were removed at specified times, centrifuged, resuspended in icecold PBS and precipitated with an equal volume of cold 10% TCA . Precipitated macromolecules were collected on glass-fibre filters and washed with 5 % TCA and ethanol . Filters were air-dried, scintillation fluid was added, and samples were counted in a liquid scintillation counter. Results were expressed as dpm [ 3 H]thymidine divided by dpm [ 14 C]thymidine for triplicate determinations . The alkali-unwinding assay was used to measure relative DNA chain elongation (Johanson and Rydberg 1977, Olive 1979) . Monolayer and 1-day-old spheroid cells were incubated for 5 min with 3 .7 kBq/ml [ 14 C]thymidine, then placed in fresh non-radioactive medium . Cells were lysed (0 . 03 M NaOH, 1 M NaCl) starting at 5 min and up to 2 h after labelling . Samples were lysed for 1 h at 4 ° C . The % DSDNA was determined for each sample, measured in triplicate .

2.6.

Topoisomerase II activity Topoisomerase II activity was measured using the trypanosome kinetoplast DNA decatenation reaction as described by Sahai and Kaplan (1986) . Kinetoplast DNA was obtained from Crithidia fasciculata provided by Dr Warren Ross, University of Florida, Gainesville . The method for isolating kDNA was also obtained from Dr Ross (personal communication) . The cells were grown in brain heart infusion medium (Difco) supplemented with 20µg/ml hemin and, in medium containing 37 kBq/ml [ 3 H]thymidine for 18h at 27 ° C, at which time the cell concentration was approximately 10 7 per ml . The cells were harvested by centrifugation, washed with PBS, and lysed in the presence of 3% sarcosyl and 1 mg/ml heat-inactivated pronase . The lysate was centrifuged through a discontinuous CsCI gradient: the bottom layer was 4 ml of a solution containing 69-6g CsCl, 1 . 8 ml 0 . 5 M EDTA, 0. 1 mg ethidium bromide, 48 m1 H 2 O, and the top layer consisted of 15 ml of a solution containing 151 . 6 g CsCl, 6 ml 0 . 5 M EDTA and 300 ml H 2 O . After a 10 min centrifugation at 20000 rpm in an SW 28 rotor, the kDNA band located between the CsCI layers was withdrawn by syringe inserted through the side of the tube, extracted with n-butanol to remove the ethidium bromide, and dialysed 18 h against two changes of 10 mm Tris, 1 mm Tris, 1 mm EDTA buffer . The solution was then centrifuged for 2 h at 27 000 rpm in the SW 28 rotor, and the



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pelleted DNA was dissolved in Tris-EDTA buffer . The specific activity of the preparation was usually about 3 x 104 dpm/µg . Chinese hamster V79 cell suspensions containing a total of 3 x 10 7 monolayer or spheroid cells were centrifuged and resuspended in 2 ml of a homogenizing medium consisting of 0 . 1 M KH 2 PO 4i pH 7 . 0, 1 mm EDTA, 1 mm 2-mercaptoethanol . The suspensions were hand-homogenized using 15 strokes at 4°C . An aliquot was removed for the determination of protein concentration using a Sigma protein assay, and the remainder of the homogenate was diluted with an equal volume of the diluting solution containing 0-02m KH 2 PO4i 0-02 mm sodium EDTA, 2 mm 2-mercaptoethanol, 10% glycerol and 1 mg/ml bovine serum albumin . The diluted solutions were dispensed in 1 ml aliquots and were frozen and stored at -25 ° C . The kDNA (0 . 25 µg) and cell homogenates (0 . 25-0 •7 5,ug/protein per reaction) were incubated in 40 pl of a reaction mixture containing 0 . 5 mm ATP, 0 . 01 M MgCl2, 0 . 06m KCI, 100µg/ml bovine serum alllumin and 0-05m Tris buffer, pH 7 . 9 . The samples were incubated at 30°C for 20 min, and the reaction was stopped by addition of 5µl 2 . 25% SDS . The extent of decatenation was linearly proportional to the amount of protein of V79 monolayer homogenates under these conditions . Enzymatically decatenated kDNA was separated from catenated kDNA by centrifugation according to the method of Sahai and Kaplan (1986) . The reaction tubes were centrifuged at 13 OOOg for 10 min and aliquots of the supernatant solutions containing decatenated DNA were assayed by liquid scintillation counting . The amount of radioactivity in the supernatant solution was compared to the total radioactivity before centrifugation, and the percentage of decatenated DNA determined. 3 . Results Etoposide was more toxic to V79 cells grown as monolayers than as spheroids . The 1-day-old spheroid survival curve demonstrated a resistant `tail' at higher etoposide doses (Figure la) as well as an increase in the size of the low-dose 1 .0 C 0

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Figure 1 . Toxicity of etoposide towards Chinese hamster V79 cells growing as monolayers or spheroids . Cells were trypsinized prior to exposure to etoposide for 1 h at 37 °C and again prior to plating to determine clongenic potential . (a) Full dose-response curves for cell grown as monolayers, for 24h in suspension culture, or after 3-7 days as spheroids ; (b) low-dose region for monolayers and 1-day-old spheroids . The mean and standard error for five independent experiments is shown . For symbols without error bars, in this and the following figures, the error is smaller than the symbol size


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shoulder region (Figure lb) . The difference in survival in the shoulder region is significant at the 99% confidence level for both the 0 . 5 and 1 µg/ml concentrations . Monolayers were exposed to 1 .3 times less etoposide than 1-day-old spheroids for the same surviving fraction of 0 . 37 . This ratio of doses for 37% survival is comparable to the ratio of 1 . 21 calculated for trypsinized monolayers and spheroids exposed, on ice, to ionizing radiation (Olive et al . 1991) . As previously shown (Durand and Goldie 1987), older spheroids containing significant fractions of noncycling cells were much more resistant to this drug . No significant differences were observed among the responses of 3-7-day-old spheroids, so results from four experiments were pooled . Two methods based on different physical principles were used to measure DNA strand breakage by etoposide . The DNA precipitation assay is based on an assay originally used to detect DNA-protein crosslinks (Smith 1962, Muller 1983, Liu et al . 1983), but which has subsequently been modified to measure DNA ssb (Olive 1988). In this method, cells are lysed in an alkaline detergent solution followed by addition of potassium chloride, heating to 65°C, and subsequent cooling on ice . The precipitate which forms contains protein and unbroken DNA, and low-speed centrifugation is used to separate this precipitate from the supernatant containing damaged DNA . Less DNA damage was measured in spheroids compared to monolayers exposed to etoposide (Figure 2a) . To reduce the amount of DNA precipitated to 37% required treating spheroids with 1 . 3 times more etoposide than monolayers . This value is in good agreement with the toxicity results, as expected, since formation of the cleavable complex has been implicated as the lethal lesion induced in the cell by epipodophyllotoxins (Glisson and Ross 1987) . In contrast, both monolayers and spheroids were equally sensitive to the topoisomerase I inhibitor, camptothecin (Figure 2b) . While the similarities between the actions of ionizing radiation and etoposide have been recognized (Smith et al . 1986), camptothecin produces only DNA ssb, not dsb (Osheroff 1989) . In addition, cell sensitivity to camptothecin is independent of proliferative status (Duguet et al . 1983) . To determine if cell cycle differences were responsible for the decrease in sensitivity of spheroids to etoposide, cell killing by another cell cycle-dependent 100 Va)

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(a) (b) Figure 2 . DNA damage induced by etoposide and camptothecin measured using the DNA precipitation assay . DNA ssb were measured in monolayers and 1-day-old spheroids immediately after a 1 h exposure to etoposide (a) or camptothecin (b). The mean± standard error for three experiments is given .

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Figure 3 . Cytotoxicity of 5-fluorouracil (5-FU) towards monolayers and 1-day-old spheroids . Cells were incubated for 2 h with 5-FU, then assayed for clonogenicity . The mean±standard error for three experiments is shown . drug, 5-fluorouracil (Heidelberger 1965), was examined . No difference was observed in the response of monolayers and 1-day-old spheroids to 5-fluorouracil (Figure 3) . The alkali unwinding assay detects the presence of DNA ssb which act as sites for DNA unwinding . The greater the number of strand breaks, the more rapid the rate of unwinding in alkali and the larger the amount of single-stranded DNA detected following neutralization . It should be noted that the alkali unwinding method and the comet assay do not involve the use of detergent which has been postulated to be a requirement for detecting strand breaks by etoposide (Glisson and Ross 1987) . The amount of DNA unwinding in alkali was reduced in 1-day-old spheroids compared to monolayers exposed to etoposide (Figure 4a), again indicating that etoposide treatment resulted in fewer strand breaks in spheroid DNA Q

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Figure 4 . DNA damage induced by etoposide measured using the alkali-unwinding assay . Monolayers (x) and one-day-old spheroids (0) were exposed for I h to etoposide before measuring DNA ssb using the alkali-unwinding assay . (a) Dose-response curves obtained after a 1 h lysis period ; (b) unwinding kinetics after exposure of cells to 0 . 5 pg/ml etoposide . Mean and standard deviation for three determinations is shown .



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compared to monolayer DNA . The kinetics of unwinding were linear for both monolayers and spheroids (Figure 4b), although previous results have shown nonlinear kinetics for spheroid cells exposed to X-rays (Olive et al. 1986) . Both the alkali unwinding assay and the DNA precipitation method measure the average DNA damage to the population . To determine if a subpopulation of the spheroid cells might be more resistant to DNA damage, as might be expected for those cells leaving the growth cycle, the comet assay was used to detect DNA strand breaks in individual cells (Olive et al . 1990b) . As with the other methods, monolayer cells exposed to 2µg/ml etoposide showed more damage than spheroid cells, but heterogeneity in response, indicated by the variance (mean divided by standard deviation) was similar for the two populations ; there was no evidence of a subpopulation of cells resistant to etoposide (Figure 5) . The decreased sensitivity of spheroid cells to etoposide could indicate either a decrease in topoisomerase II activity, or a decrease in the ability of etoposide to interact with DNA and/or the enzyme . Therefore, topoisomerase II activity in extracts of monolayer and spheroid cells was measured using a decatenation reaction . As shown in Table 1, topoisomerase II activity of spheroids was significantly reduced compared to that of monolayers . Spheroids 3-4 days old showed a greater decrease in topoisomerase II activity indicative of a decrease in growth fraction . The decrease in topoisomerase II activity in 1-day-old spheroids could indicate a decrease in the growth fraction in these spheroids, or perhaps a decrease in the fraction of cells synthesizing DNA (Chow and Ross 1987, Sullivan et al . 1986) . Although previous results have shown no change in cell cycle time for 1-day-old

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Tail Moment Figure 5 . DNA damage in monolayers and 1-day-old spheroid cells detected using the comet assay . DNA damage induced by 2 pg/ml etoposide was measured in 200 individual cells . The tail moment is an indication of the amount of DNA damage produced by this drug and is linearly related to dose of etoposide (Olive et al . 1990b) . The average tail moment for the spheroids was 15-3 and for the monolayers, 24-1 ; comparable value for untreated cells were 2 . 21 for the monolayers and 2-4 for the spheroids . Untreated cells produced tail moments which fell (95% of the time) below the value indicated by the vertical line .

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Table 1 . Topo II activity in monolayers and spheroids measured by decatenation of kinetoplast DNA (%DNA decatenation per 0 . 5 pg protein) . Monolayers One-day-old spheroids Three- to four-day-old spheroids

71 . 7, 66 . 6, 62 (100%) 37 . 9, 53 . 5, 44 . 3 (68%) 16-8,26-5 (32 .5%)


Values shown are for separate experiments and are the averages of two or three replicates each . The 1-day-old spheroids show significantly less topoisomerase II activity than monolayers at the 95% confidence level using a paired t-test .

spheroids versus monolayers (Durand 1976, Olive 1989), the possibility that DNA replication rate might differ in monolayers and small spheroids was re-examined in light of the observation that newly synthesized DNA is associated with topoisomerase II (Nelson et al . 1986) . To examine the rate of DNA synthesis in monolayers and spheroids, the rate of incorporation of [3 H]thymidine was measured . In addition, the rate of DNA chain elongation was determined by applying the alkaliunwinding assay at various times after a pulse of [ 3 H]thymidine (Olive 1978) . When actively growing cells are incubated briefly with [ 3 H]thymidine, the radiolabel first appears in the single-stranded DNA eluted from the hydroxyapatite column, but as replication proceeds beyond the labelled region, the radiolabelled material is found increasingly in the native double-stranded form (Johanson and Rydberg 1977) . Both incorporation of thymidine into replicons and elongation rates from initiated replicons were identical for cells from monolayers and one-day-old spheroids (Figure 6) .

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Figure 6 . DNA initiation and elongation in V79 monolayers (x) and 1-day-old spheroids (0) . (a) Incorporation of [ 3H] thymidine was measured in cells uniformly labelled with [ 14 C]thymidine prior to growth for 24h as monolayers or spheroids . Cells were then placed in medium containing 37 kBq/ml [3 H]thymidine for up to 2 h . Cells were washed, and radioactivity incorporated into macromolecules was precipitated on glass-fibre filters using 5% TCA . The ratio 3 H to 14 C was determined using liquid scintillation counting . (b) Monolayers and spheroids were incubated for 5 min with medium containing 74 kBq/ml [ 3 H]thymidine . At various times after labelling, cells were lysed in alkali and examined for rate of elongation of DNA using the alkaliunwinding assay . The mean and standard error for three determinations is shown .

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4.


Discussion The most obvious explanation for the - 30% decrease in etoposide sensitivity and topoisomerase II activity of 1-day-old spheroids would be a decrease in their growth fraction and/or a decrease in the fraction of cells in S phase . One-week-old spheroids contain 50-60% non-cycling cells, and these cells are 50-100 times more resistant to etoposide than cycling cells (Durand and Goldie 1987) . However, this does not appear to be the explanation for our results using 1-day-old spheroids . First, previous studies indicate that there is no change in distribution of cells in the growth cycle when cells are grown as spheroids for only 24 h (Durand 1976, Olive 1989) . In addition, the rate of uptake of [3H]thymidine by monolayers and 24-h spheroids was identical, as was the rate of chain elongation of newly synthesized DNA (Figure 5) . There was no evidence of a population of cells resistant to etoposide as indicated by the comet assay (Figure 4) . If 30% of the spheroid cells are out of cycle (a reasonable assumption if topoisomerase II activity is reduced by 30%), this non-cycling population would have been easily detected by the comet assay, or by cell survival assays . In larger spheroids the population of non-cycling cells, resistant to etoposide, is clearly identified using the comet assay (Olive et al. 1990b). In addition, monolayers and spheroids were equally sensitive to killing by 5-fluorouracil, another drug preferentially toxic to actively cycling cells . Lastly, extrapolating the resistant `tail' on the survival curve in Figure 1(a) back to the ordinate on this graph indicates clearly that no more than 1 % of the cells are noncycling . The difference in initial DNA damage detected in Figure 2(a) apparently reflects the difference in the shoulder on the survival curve, not the resistant tail (Figure 2b) . However, while these results argue against a change in growth fraction being responsible for the increased resistance of spheroids to etoposide, we cannot rule out the possibility that the decrease in topoisomerase I I activity (Table 1) is an early indication of a change in growth fraction . Reduction in decatenation activity is likely to occur before cells have actually left the cell cycle, and this decrease in activity should also be reflected in a decrease in sensitivity to killing and DNA damage by etoposide . However, this hypothesis is not consistent with the change in the shoulder region of the survival curve which reflects a change in all of the cells of the spheroids, not just those on the inside of these small clusters which are destined to leave the cell cycle . If a decrease in growth fraction is not the explanation for the resistance of 1-dayold spheroids to etoposide, then there are several other possibilities . It has been suggested that changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II (Smith and Mackinson 1989) . This hypothesis is appealing, since previous results suggest that the conformation or packaging of DNA may be altered in spheroid cells (Olive et al . 1986, Gordon et al . 1990) . Other examples of the influence of chromatin conformation on etoposide toxicity include the effects of tumour-promoting phorbol esters, polyamines, and intracellular ionic environment (Zwelling et al. 1988, Lawrence et al . 1989) . The observed decrease in topoisomerase II activity in extracts from spheroids compared to monolayer cells would tend to argue against a change in chromatin structure as being responsible for the decrease in etoposide sensitivity in spheroids, unless this change in chromatin structure were : (1) accompanied by a decrease in growth fraction, or (2) dictating the decrease in topoisomerase II activity . The resistance of irradiated spheroids to alkali unwinding (Olive et al. 1986) and the apparent greater



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stability of spheroid nuclei to supercoil relaxation or detergent-induced degradation (Gordon et al . 1990) support this second possibility . We therefore suggest that less topoisomerase II may be required to establish or maintain the condensed chromosome architecture in spheroid nuclei . Another possibility is that there is a decrease in the amount of topoisomerase II in spheroid cells which is unrelated to growth fraction or DNA conformation, and a final explanation is that the enzyme has undergone a change which alters drugenzyme-DNA binding . These hypotheses seem less likely than the previous one, since we are not examining mutant cells with different genetic backgrounds, but the same cell grown in two different environments . Chinese hamster V79 cells grown in suspension culture will revert back to monolayer characteristics in less than one cell doubling after return to tissue culture dishes (Olive and Durand 1985) . Quantification of topoisomerase II using immunoblotting is necessary to distinguish between a decrease in the amount of enzyme versus a decrease in enzyme activity ; however, enzyme activity, not amount, generally correlates better with sensitivity to etoposide (Singh and Lavin 1989, Glisson and Ross 1987) . One-day-old spheroids have previously been shown to be more resistant than monolayers to killing by adriamycin (Durand 1981) and heat (Dobrucki and Bleehen 1985, Durand 1978, Wigle and Sutherland 1985) . Interestingly, topoisomerase II has been implicated in the toxicity of both of these agents (Tewey et al . 1984, Warters and Brizgys 1988) . The decrease in topoisomerase I I activity in spheroids could help to explain their resistance to these agents . As previously observed for cells treated with X-rays and etoposide, the increased survival in spheroids treated with adriamycin and heat is a result of an increase in the shoulder on the survival curve . Since spheroids were disaggregated prior to treatment wth these agents, cell-cell contact at the time of treatment is not necessary to observe resistance . The mechanism responsible for the increased radiation resistance of 1-day-old spheroids compared to monolayers is not known, although there is a general consensus that spheroids repair radiation-induced DNA damage more effectively than monolayers . The role of topoisomerase II in DNA repair is not established, yet it seems clear that this enzyme could be involved in dsb repair and recombination-type events . In addition, topoisomerase II is an important structural component of the nuclear matrix, and evidence suggests that it is involved in maintaining the structure of the DNA loops . Evidence from other studies suggests that the loop size is not altered in spheroid cells (Olive et al . 1986), yet the stability of these loops appears to be increased (Gordon et al . 1990), as though the link between DNA and the nuclear matrix were more stable in spheroids . It is possible that topoisomerase II is decreased in 1-day-old spheroids because less of this enzyme is required by these cells and/or because other proteins, synthesized only in spheroids, now aid in maintaining loop stability . In summary, our results show that Chinese hamster V79 spheroids are less sensitive than monolayers to cell killing and DNA damage by etoposide . This decrease in sensitivity is consistent with the small but significant decrease in topoisomerase II activity in 1-day-old spheroids . A conclusion which is consistent with these data is that topoisomerase II is reduced in spheroid cells for reasons other than a change in growth fraction . A possible explanation is that the change in DNA conformation which occurs when cells are grown as spheroids reduces their requirement for this enzyme .

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et al .

Acknowledgements This work was supported by the National Cancer Institute of Canada, the Medical Research Council of Canada and by NCI grant R37-CA-15901 from the US Public Health Service .


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WANG, J . C ., WARTERS, R .

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line.

Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells.

Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells.

Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells.

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells.

Structure-activity study of paracetamol analogues: inhibition of replicative DNA synthesis in V79 Chinese hamster cells.

Mutagenicity of vinyl chloride, vinylidene chloride and chloroprene in V79 Chinese hamster cells.

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells.

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

Interaction of amosite and surface-modified amosite with a V79-4 (Chinese hamster lung) cell line.

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

Effect of tumor promoting agents on mutation frequencies in cultured V79 Chinese hamster cells.

Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells.

Induction of micronuclei in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol.

Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids.

Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells.

Changes in cell cycle distribution of V79 Chinese hamster fibroblasts after irradiation at different wavelengths.

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol.

Effect of alkane tumor-promoting agents on chemically induced mutagenesis in cultured V79 Chinese hamster cells.

Mechanisms involved in resistance of preirradiated Chinese hamster V79 cells to cytotoxic drugs are multifactoral.

Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates.