Novobiocin, Nalidixic Acid, Etoposide, and 4'-(9-Acridinylamino)Methanesulfon-M- Anisidide Effects on G2 and Mitotic Chinese Hamster Ovary Cell Progression1

Home , Nalidixic acid, Novobiocin

(CANCER RESEARCH 49. 4752-4757. September 1. 1989] Novobiocin, Nalidixic Acid, Etoposide, and 4'-(9-Acridinylamino)methanesulfon-m- anisidide Effects on G2 and Mitotic Chinese Hamster Ovary Cell Progression1

Roy Rowley2 and Lila Kort

Experimental Oncology, Department of Radiology, University of Utah Medical Center, Salt Lake City, Utah 84132

ABSTRACT not necessarily coincide with changes in the levels of assayable topoisomerase II antigen (12, 13). Exponentially growing Chinese hamster ovary cells, exposed to inhib One approach to identifying topoisomerase H-mediated proc itors of topoisomerase II (novobiocin, nalidixic acid, etoposide, and 4'- esses in the mammalian cell cycle is to locate the point(s) in (9-acridinylamino)methanesulfon-m-anisidide were blocked in progres sion through (.-. The manner of recovery from the novobiocin-induced the cell cycle at which known inhibitors of topoisomerase II block cell progress. We have conducted such an investigation, block, following drug removal, indicated that the blockade was at and concentrating on the latter end of the CHO1 cell cycle. A variety before a specific point in ( ;. (a transition point). The transition point for novobiocin and putative transition points for nalidixic acid and 4'-(9- of inhibitors of bacterial DNA gyrase have been identified, e.g., acridinylamino)methanesulfon-m-anisidide were about 30 min before novobiocin, coumermycin A, (14), and nalidixic acid (15) which metaphase. The transition point for nalidixic acid varied with concentra also show inhibition of the eukaryotic enzyme, albeit at higher tion from about 70 min before metaphase, at 1 fig/ml, to 24 min before concentrations (Ref. 16 and references therein), and may be metaphase at 15 Mg/mland above. The novobiocin- and nalidixic acid- used for this purpose. Clearly, however, such inhibitors are not induced (. block could not be accounted for by cytotoxicity or DNA entirely specific for topoisomerase II (17) and, as pointed out damage (detected by neutral elution). The novobiocin-induced G2 block by Charcosset (18), have side effects that might also result in could not be attributed to gross RNA synthesis inhibition. Progress G2 arrest. For example, the topoisomerase-binding acridine, beyond metaphase was blocked by novobiocin but not by nalidixic acid, when cells were exposed to drug concentrations which inhibited (... cell mAMSA, produces double stranded breaks in the DNA (19), produces chromosome aberrations (20), and inhibits RNA syn progression. It is suggested that the progression of Chinese hamster thesis,4 any one of which may cause G2 arrest (21-23). We have ovary cells into but not through mitosis may require topoisomerase II. therefore used four known topoisomerase II inhibitors with different modes of action and compared their influence on G2 INTRODUCTION and mitotic cell progression with their abilities to produce cell killing, to induce DNA double strand breaks, and to inhibit Type II topoisomerases catalyze both the induction and re RNA synthesis. The agents tested were novobiocin, VP-16, m- joining of double-strand breaks in DNA. This action makes AMSA, and nalidixic acid. Novobiocin has been shown to bind possible relaxation of supercoiled DNA, DNA catenation, de to the ATP-binding site on the B subunit of Escherichia coli catenation, knotting, and unknotting (1-3). A bacterial version gyrase (14), blocking the ATP-requiring activities of the enzyme of this enzyme, DNA gyrase, has been implicated in replication, repair, recombination, and transcription (1-3); however, in (1). Nalidixic acid binds to the A subunit (15), the portion of the enzyme responsible for DNA nicking and closing activities. eukaryotes, only one critical function has been attributed to m-AMSA is believed to stabilize the binding of the enzyme to topoisomerase II, decatenation of replicated DNA at mitosis. the DNA, resulting in an inactive complex that may be proc This was demonstrated by inactivation of topoisomerase II in essed to form a DNA double strand break (19). VP-16 also the temperature-sensitive Saccharomyces cerevisiae mutant, top binds the enzyme but unlike m-AMSA does not also intercalate Â¡I.Following movement to the restrictive temperature, cells with DNA (24). underwent only one round of replicative DNA synthesis and became blocked in medial nuclear division. Plasmids in the arrested cells were in the form of multiply intertwined, ca MATERIALS AND METHODS tenated dimers (4, 5) and the yeast chromosomes showed an CHO Cells. CHO cells were obtained from the American Type elevated rate of nondisjunction (6), consistent with failure to Culture Collection (Rockville, MD). The cells were maintained as disentangle sister chromatids. In similar experiments with a exponentially growing monolayers (doubling time, approximately 12 cold-sensitive mutant of Schizosaccharomyces pombe, topoi h) in plastic flasks (Falcon Plastics) containing McCoy's Medium 5A somerase activity was apparently required for both mitotic (GIBCO) supplemented with 10% fetal bovine serum (Hazelton, Den chromosome condensation and segregation (7). While no like ver, PA). Growth conditions were: temperature, 37Â°C;humidity, 85- role has been clearly demonstrated outside of yeast cells, it is 100%; atmosphere, 6% CO2/94% air. The cells were periodically eval consistent with the location of topoisomerase II in the Droso- uated for Mycoplasma contamination by the uridine/uracil incorpora phila nuclear matrix (8) and in the mammalian metaphase tion test. chromosome scaffold, at the foot of the radial loop (9, 10). A Mitotic Shake-off Procedure (Mitotic Cell Selection). Cell progression to mitosis was monitored by mitotic cell selection as described previ role for topoisomerase II in mammalian cell mitosis is also ously (25). T-75 flasks containing approximately IO7 cells were suggested by the marked sensitivity of mammalian cells in (11) mounted on an Eberbach reciprocating shaker housed in a modified or just prior to (12) mitosis to DNA cleavage by drugs believed CO2 incubator. The flasks were shaken for 20 s every 10 min, the to bind the enzyme. Note, however, that cell cycle-related medium and detached cells were decanted, and the medium was re changes in the susceptibility of a cell to such DNA cleavage do placed. The number of detached cells was determined by Coulter Counter (Hialeah, FL) and their size frequency-distribution was moni Received 7/15/88; revised 2/14/89. 4/25/89; accepted 5/25/89. The costs of publication of this article were defrayed in part by the payment tored by Coulter Channelyzer to ensure that the cells were mitotic (i.e., of page charges. This article must therefore be hereby marked advertisement in exhibited a normal distribution with a peak at approximately twice the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation was supported by USPHS Grant CA 40245 awarded by 3The abbreviations used are: CHO. Chinese hamster ovary; m-AMSA, 4'-(9- the National Cancer Institute. Department of Health and Human Services. acridinylamino)methanesulfon-m-anisidide; VP-16, etoposide. 2To whom requests for reprints should be addressed. 4 R. Rowley, unpublished observations. 4752

size of the peak for asynchronous cells). The true mitotic index was determined by light microscopy of fixed, selected cells and was normally >97%. Drugs were administered to the cells by substituting drug- containing medium for control medium during refeeding of the monolayers. Novobiocin (Sigma) and actinomycin D (Sigma) were dissolved directly in medium. Nalidixic acid (Sigma) was dissolved as a concen trate in sodium hydroxide, before dilution into medium. m-AMSA (Pharmaceutical Resources Branch, National Cancer Institute) was dissolved as a concentrate in dimethyl sulfoxide (5 mg/ml) before dilution in medium. VP-16 (Bristol-Myers) was diluted from an inject able drug/saline concentrate. The solvents alone were without influence on cell progression. Mitotic cell selection data were interpreted as described by Schneiderman et al. (25) and Kimler et al. (26). Survival Assays. Cells were plated into T-25 plastic flasks (Corning) at appropriate density and maintained under normal culture conditions in medium containing penicillin and streptomycin. Clones formed after 7 days of incubation were fixed and stained with crystal violet in methanol and counted if >50 cells. DNA Damage Detection. DNA double stranded breaks were detected by neutral filter elution (27, 28), but using lysis and elution buffers at pH 7.2, as described previously (29). Exponentially growing CHO cells were labeled overnight with ['"CJthymidine at 0.1 Â¿iCi/ml(50 Ci/mol). The cells, 2 x IO6 in ice-cold phosphate-buffered saline, were then loaded onto 47-mm, l.Q-pm porosity polycarbonate filters and rinsed with cold phosphate-buffered saline. Lysis solution was added [0.5 mg/ ml proteinase K in elution buffer (2% w/v sodium dodecyl sulfate, 50 HIMTris-HCl, 50 HIMglycine, and 20 mivisodium EDTA, pH 7.2)] and 50 MINUTES this solution was pumped past the filter for l h at room temperature at a rate of 0.045 ml/min. Elution buffer was then added and the pumping Fig. 1. A, number of mitotic cells detached from a cell monolayer (normalized was continued. The eluate was collected as 4.5 nil sequential fractions, to an untreated control) plotted against time after exposure to novobiocin (300 the fraction volumes were measured, and 1-ml samples were neutralized Mg/ml. A: 500 fig/ml. O; 750 Mg/ml, D; 1000 Mg/ml. â€¢¿.allcontinuously from and mixed with scintillation fluid for scintillation counting. At the end time zero). B, similar data for nalidixic acid (0.5 mg/ml. A; 0.75 mg/ml. O; l mg/ml, D; 1.25 mg/ml, â€¢¿). of the experiment, the radioactivity remaining on the filter, in the lysis solution, and in the final apparatus rinse (0.5 M NaOH) was similarly assayed. The total radioactivity per treatment was estimated by sum ming the total counts on the filter, in the lysis and rinsing solutions, and in every fraction. The counts (DNA) remaining on the filter after the nth fraction had passed were calculated, knowing the total counts per treatment and the sum of the counts per fraction for fractions 1 to n. The proportion of counts remaining on the filter after each fraction was then plotted against the total volume eluted up to that fraction.

RESULTS AND DISCUSSION (â€¢Block by Topoisomerase II Inhibitors. The mitotic cell selection technique allows the number of cells entering mitosis to be determined at 10-min intervals following treatment of a monolayer cell population. A slowing or block in cell progres sion is thus detected as a decrease in the number of mitotic cells. Figs. 1 and 2 show the effect of novobiocin (300, 500, 750, or 1000 Mg/ml), nalidixic acid (500, 750, 1000, or 1250 Mg/ml), m-AMSA (0.1, 0.3, and 0.4 Mg/ml) and VP-16 (1, 2.5, 5, and 15 Mg/ml) on CHO cell progression to mitosis, as monitored by mitotic cell selection (cell numbers are given relative to controls run in parallel). In all cases the degree of inhibition of cell entry to mitosis increased with drug concen tration, such that the lowest tested concentration to reduce the number of mitotic cells to zero (or near zero) was 750 Mg/ml for novobiocin, 1 mg/ml for nalidixic acid, 0.4 Mg/ml for m- AMSA, and 2.5 Mg/m' for VP-16. Higher concentrations of MINUTES Fig. 2. .I. number of mitotic cells detached from a cell monolayer (normalized novobiocin or nalidixic acid did not significantly alter these to an untreated control) plotted against time after exposure to m-AMSA (0.1 Mg/ kinetics and the block produced by novobiocin was leaky in that ml. A; 0.3 Mg/ml. O; 0.4 Mg/ml, D). B, similar data for VP-16 (1 Mg/ml. A; 2.5 the yield of mitotic cells did not drop to zero at any concentra Mg/ml, O; 5 Mg/ml. D; 15 Mg/ml, â€¢¿). tion used. The time required for the number of cells entering mitosis to in minutes, for the same number of control cells to enter fall to zero following application of a given concentration of mitosis. This value is equal to the area under the curve for the drug is given in Table 1. Values were calculated relative to plots of mitotic cell number versus time after drug addition controls run in parallel and are therefore expressed as the time, (Figs. 1 and 2). These times were approximately the same for 4753

Table 1 Transition points in the GÃ¬phaseof the CHO cell cycle 1.6-1 AgentNovobiocin transitionSE*31 point" Â± Mg/ml)Nalidixic(750 Â±230 mg/ml)m-AMSAacid (1 Â±339 Mg/ml)VP-16(see(0.4 Â±164 3)CycloheximideFig. Mg/ml)Actinomycin(50 Â±290 D2 fig/ml5 Â±777 Mg/mllO^g/ml1 Â±356 Â±52535 5Mg/ml20 fig/ml25 Â±331312830 fig/ml30 Mg/ml40 /ig/ml50 Mg/mlMean " Minutes before the earliest point of cell detachment in mitosis. * Standard error of the mean of at least 3 assays. Transition point values without errors are single assays. Fig. 4. Number of mitotic cells detached from a cell monolayer (normalized to an untreated control) plotted against time after the start of exposure to 500 Mg/ml novobiocin. Novobiocin was removed after 60 min (NOVO OFF). Data are means for 3 separate experiments Â±SEM (bars).

including an increase in the rate of cell entry to mitosis that transiently exceeded the control. The speed of recovery [roughly the same rate at which cell entry to mitosis declines following

10- X-irradiation (25)] is consistent with cell movement from a well defined point of arrest, and the overshoot is consistent with an accumulation of cells at that point. The arrest and recovery data, taken together, thus suggest that there exists in G2 a point beyond which the cells are refractory to the cytostatic effects of the drug, i.e., a transition point. As with higher doses of X- 10.0 irradiation (26), recovery after higher doses of novobiocin is Fig. 3. Time required for the number of mitotic cells detached from a cell less rapid (Fig. 6), possibly reflecting additional modes of action monolayer to fall to zero following exposure to different concentrations of VP- only evident at such concentrations. The position of the tran 16. Each point represents the area under the curve of plots such as those in Fig. 2B. See text for details. sition point for novobiocin is similar to that for ellipticine in CHO cells [40 min before mitosis (31)] and for CI921, an novobiocin, nalidixic acid, and m-AMSA. m-AMSA inhibited amsacrine derivative used on CHO cells [30 min before mitosis (32)]. These data also confirm other reports of G2 arrest induc progression slightly sooner at 0.5 /Â¿g/ml,but this concentration tion by topoisomerase II directed chemotherapeutic agents, e.g., sometimes caused detachment of interphase cells. Following TV-trifluoracetyladriamycin-M-valerate (33), m-AMSA (34), VP-16, the time required to halt progress to mitosis was con and a variety of ellipticine derivatives (35). centration dependent (Fig. 3). The plot reached a plateau at 24 The existence of a transition point for G2 cell progression min before metaphase (15 ng/ml and above). inhibition by VP-16 (between 2.5 and 5 Mg/ml) is more readily The continued progression of cells after the addition of drug may be interpreted in two ways. Either cell progression is evident. Cell progression following drug addition is initially blocked at a specific point in G2 (dependent upon concentration like the control and then drops off rapidly (Fig. 2), as expected in the case of VP-16) and cells beyond this point are refractory for a specific progression block. However, for concentrations or progression inhibition is nonspecific in the position of the above 5 Mg/ml, nonspecific inhibition of progression may also block point and the continued progression of late G2 cells into occur, inasmuch as progression inhibition is evident immedi ately after drug addition. A concentration-dependent movement mitosis following drug addition simply reflects the time re quired for the drug to enter the cell. The former interpretation of the transition point has also been seen after actinomycin D is applied to the action of radiation on G2 cell progression (25), treatment of CHO cells (36), where it was attributed to multiple where the point of arrest is termed the transition point. It has drug effects [RNA synthesis inhibition and direct inhibition of also been applied to the action of protein and RNA synthesis chromosome condensation (36)]. We note that actinomycin D inhibitors (30). is also postulated to inhibit topoisomerase II (37). We suggest that the transition point interpretation is appli The position of a transition point in the cell cycle is believed cable here but that the existence of clearly defined transition to reflect the mechanism of action of the blocking agent (30, points (i.e., continued posttreatment progression followed by a 38), marking the last execution point of the process that the sharp decrease to zero in mitotic cell number) for novobiocin, blocking agent inhibits. The transition point for novobiocin and nalidixic acid, and m-AMSA may be masked by additional, presumed transition points for nalidixic acid and m-AMSA nonspecific drug effects on progression. Justification for this (Table 1) are approximately 30 min before the point in mitosis interpretation is provided in Fig. 4. Cells were exposed to 500 at which the cells first become detachable by the shakeoff Mg/ml novobiocin for l h and their rate of entry to mitosis procedure [approximately metaphase (25)]. monitored during and after exposure. Novobiocin blocked prog RNA Synthesis Inhibition. The possibility that topoisomerase ress into mitosis as described above. Drug removal was followed II inhibitors block G2 cell progression by inhibition of RNA first by continued inhibition of entry to mitosis, lasting approx synthesis was addressed, for novobiocin only, by comparison imately 50 min, then by a rapid resumption of cell progress, with the effects on G2 cell progression of actinomycin D, a 4754

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. TOPOISOMERASE II INHIBITORS AND G2 ARREST potent RNA synthesis inhibitor. Fig. 5 shows the concentration novobiocin-induced inhibition of progress through G2 began dependencies for inhibition of ['HJuridine incorporation by approximately l h after novobiocin removal, in the presence or novobiocin and by actinomycin D. Uridine incorporation in the absence of actinomycin D, indicating that the novobiocin-in presence of novobiocin was about 10% of control between 500 duced block was not a result of gross RNA synthesis inhibition. and 1000 Mg/ml, also approximately the concentration to cause The population recovering in the presence of actinomycin D maximal suppression of cell entry to mitosis. Uridine incorpo was smaller than the segment of the cell cycle that lies between ration in the presence of actinomycin D was <10% for concen the actinomycin D transition point and the novobiocin transi trations above 5 ng/m\. At 5 Â¿tg/ml,actinomycin D blocked G2 tion point (approximately the equivalent of the yield from 2.03 cell progression with an average transition point at 77 min control shakes, compared with an available population of 6.4 before the metaphase, or approximately 40 min before the control shake equivalents). While the reason for this is not transition point for novobiocin (Table 1). The mechanism of known, it seems possible that the delay induced by novobiocin induction of G2 arrest by these two drugs must therefore differ was of sufficient duration to allow decay of a fraction of the at these concentrations. It is recognized that actinomycin D is mRNA species required for progression through G:. Cells thus also a putative topoisomerase II inhibitor (37) and that at high affected would then be susceptible to actinomycin D even concentrations the G2 transition point for actinomycin D co though beyond the transition point for this actinomycin D incides with that for the other topoisomerase II inhibitors concentration. This explanation was previously proposed for (Table 1; Ref. 36). the acquisition of susceptibility of X-irradiated cells to inhibi To demonstrate further that the novobiocin-induced block to tion of recovery from arrest by actinomycin D with increasing G2 cell progression was not due to inhibition of RNA synthesis, duration of X-ray-induced arrest (23). recovery from novobiocin-induced arrest was examined with or Cell Killing and DNA Damage Induction. It is well docu without concomitant RNA synthesis inhibition (>95% inhibi mented (e.g., Ref. 22) that agents with clastogenic and cytotoxic tion of uridine incorporation). Fig. 6 shows the effect on cell properties frequently induce G2 arrest. VP-16 and m-AMSA progression to mitosis of (a) 750 ng/m\ novobiocin added at are chemotherapeutic agents with marked cytotoxicity (11, 39) time zero and removed at 60 min, (Â¿>)750Mg/ml novobiocin and may clearly work in this way. To test novobiocin and added at time zero and replaced at 60 min with 5 Mg/ml nalidixic acid for this mode of action, exponentially growing actinomycin D, and (c) 5 Mg/ml actinomycin D alone added at CHO cells were exposed to novobiocin (750 Mg/ml) or nalidixic 60 min. The transition points for these treatments were 26 min acid (1 mg/ml) for periods up to 4 h, the drug was washed out, for novobiocin and 90 min for actinomycin D. Recovery from and then the cells were trypsinized and tested for clone-forming ability. Untreated cells had a plating efficiency of approximately 80%. No significant decrease in survival was observed after either drug (Fig. 7). The lack of effect on cell viability excludes drug-induced cytotoxicity as a cause of G2 arrest for novobiocin and nalidixic acid; however, assays were also made for DNA damage induction. Exponentially growing CHO cells were ex posed for l h to the same drugs at the same concentrations used for the cell killing studies and then immediately tested for

400 600 (ig/ml o 1 Fig. 5. Level of [3H]uridine incorporation (normalized to an untreated control) plotted against drug concentration for cells exposed to novobiocin (O) or actino mycin D (â€¢).

Act-D ON

0.01-

o.ooi-

200 o oooi Fig. 6. Number of mitotic cells detached from a cell monolayer (normalized to an untreated control) plotted against time after the beginning of treatment. Cells were exposed to the drugs as indicated. Novobiocin (750 jig/ml) was present Fig. 7. Surviving fractions (log scale) of CHO cells exposed to novobiocin from zero to 60 min (NOVO OFF). Actinomycin D (Act-D) was present from 60 (750 (ig/ml, O), nalidixic acid (1 mg/ml. A), or m-AMSA (0.4 Mg/ml. D) for min onwards. periods up to 4 h. Bars, where larger than the symbol, represent Â±SEM. 4755

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. TOPOISOMERASE I] INHIBITORS AND G2 ARREST 100- the presence of DNA damage by neutral filter elution. This assay is sensitive to the presence of DNA double strand breaks, 80- a suggested cause of G2 arrest following exposure to ionizing radiation (21). Novobiocin and nalidixic acid did not alter the 60- rate of DNA elution (Fig. 8). m-AMSA and VP-16 both induce DNA strand breaks (Fig. 8; Refs. 19 and 39). 40- Inhibitor Effects on the Completion of Mitosis. Experiments 20- were performed to determine whether topoisomerase II inhibi tors blocked progression at other points in mitosis. Exponen 0 tially growing CHO cell monolayers were exposed to a drug 0 10 20 30 40 and then shaken at 10-min intervals as above. The products of MINUTES the third shake after treatment were then collected, transferred Fig. 11. Percentage of nalidixic acid treated CHO cells in metaphase (O), in anaphase (D), or having entered telophase (A) plotted against time after cell to a prewarmed spinner flask, and incubated under normal detachment from a monolayer by mitotic cell selection. The cells were exposed growth conditions in the continued presence of the drug. The to l mg/ml nalidixic acid for 30 min prior to detachment. progress of these cells through mitosis was monitored by mi croscopic observation of samples withdrawn at 3-min intervals. proportion of cells in anaphase was observed at approximately Untreated cells (Fig. 9) completed metaphase by approximately 10 min after detachment and anaphase was largely completed 15 min after detachment from the monolayer. The highest by 20 min after detachment. Binucleate cells without individ ually discernible chromatids (scored as telophase cells) and cells

o '-O . .. ,...,,...... ,.o^^"Q~â€” with a single nucleus showing varying degrees of chromatin Ã•Ã•0.9-t decondensation (possibly ruptured telophase cells or cells that Â°8- had completed division) accumulated from 5 to 10 min post- rr< 0.7-Qu. detachment onwards. Virtually all cells had entered or passed ~.~~~~o telophase by approximately 30 min postdetachment. In the 0.6- oÂ¡0.5-Ua:_. presence of novobiocin (750 Â¿/g/ml),the majority of cells re a tained a metaphase morphology for at least 48 min after de tachment (Fig. 10). The block to progression was somewhat leaky, however, because a small percentage of anaphase and

FRACTIONS telophase/divided cells was also observed throughout the sam Fig. 8. Fraction of DNA retained on the filter (log scale) plotted against time pling period. Nalidixic acid (1 mg/ml) did not block progress after the beginning of elution for DNA from untreated cells (â€¢),cellsexposed to through mitosis (Fig. 11), although completion of metaphase novobiocin (750 Â¿ig/ml.O), nalidixic acid (1 mg/ml, A), or m-AMSA (0.4 fig/ml, D) for l h immediately prior to lysis and filter elution at pH 7.2. was prolonged and the proportion of cells in anaphase at any time point consequently less than the control. Cycloheximide 120-100-80-60-40-20-n/*/AD-Q (50 Mg/ml) was also without effect on the completion of mitosis (data not shown). If novobiocin and nalidixic acid both inhibit topoisomerase II activity in this cell system, then the metaphase block, induced only by novobiocin, cannot be due to inhibition of topoisomerase II. Only one block in progression from G2 /K through mitosis may be attributed to an effect on topoisomerase 11activity. /\Jx Conclusions. The findings indicate that the block in G cell Â°x^>s^, progression induced by novobiocin was not secondary to inhi D Â°"~~~0â€”n 0- bition of RNA synthesis, the presence of DNA double strand breaks, or cell killing. It is therefore possible that novobiocin 0 10 MINUTES blocked progress by inactivation of a necessary step in prepa Fig. 9. Percentage of untreated CHO cells in metaphase (O), in anaphase (D), rations for mitosis. The microscopic appearance of the CHO or having entered telophase (A) plotted against time after cell detachment from a cell nucleus in cells arrested at the X-ray transition point has monolayer by mitotic cell selection. been examined by Carlson (40). The arrest point approximates 100-, to the earliest time at which chromosome condensation can be 0-0â€”-0,0 o detected. Chromosome condensation may thus be the step 80- susceptible to novobiocin, possibly mediated by inhibition of topoisomerase II. The similar action of nalidixic acid, m-AMSA 60- and VP-16 on G2 cell progression is consistent with this inter 40- pretation. It was shown recently (6) that in S. cerevisiae, an active mitotic spindle must be present to allow topoisomerase II to function completely: topoisomerase II disengages sister chro matids; the spindle pulls them apart. If our own interpretation is correct and topoisomerase II in CHO cells is required only Fig. 10. Percentage of novobiocin-treated CHO cells in metaphase (O), in at the G2-M boundary (approximately 30 min before anaphase), anaphase (D), or having entered telophase (A) plotted against time after cell detachment from a monolayer by mitotic cell selection. The cells were exposed then the enzyme in these cells can complete its function before to 750 Mg/ml novobiocin for 30 min prior to detachment. a spindle has formed. 4756

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. Novobiocin, Nalidixic Acid, Etoposide, and 4′ -(9-Acridinylamino)methanesulfon-m-anisidide Effects on G2 and Mitotic Chinese Hamster Ovary Cell Progression

Roy Rowley and Lila Kort

Cancer Res 1989;49:4752-4757.

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