[CANCER RESEARCH 46, 4032-4040, August 1986]

Mitomycin-induced Chromatid Breaks in HeLa Cells: A Consequence of IncompleteDNA Replication1

Marguerite A. Sognier2 and Walter N. Hittelman3

Department of Chemotherapy Research, the university of Texas M. D. Anderson Hospital and Tumor Institute at Houston, and the University of Texas Graduate Schoolof BiomédicalSciences, Houston Texas 77030

ABSTRACT

The formation of chromosome aberrations induced by alkylating agentssuch as mitomycin C has been shown to require the passage of the treatedcell through S phase. However, the exact mechanisms by which mito-mycin C-induced DNA lesions are translated into chromosome aberrations during S phase are not known. The purpose of these studies was tobetter understand the molecular basis of chromosome aberration formation after mitomycin C treatment. The morphology of metaphases of cellstreated in <., phase with mitomycin C resembled that of prematurelycondensed chromosomes of S-phase cells. Consequently we postulatedthat chromosome aberrations resulted from cells reaching mitosis withoutcompleting DNA replication. This was tested by treating HeLa cells in<., phase with mitomycin C and then analyzing these cells at mitosis forresidual DNA damage and DNA content. Utilizing the DNA alkalineelution assay for DNA damage, we showed that HeLa cells progressthrough S phase into mitosis with intact DNA-DNA interstrand crosslinks. These cross-links, originally induced into parental DNA, wereassociated equally with parental and newly replicated DNA at the timethe cells reached mitosis. This suggests that recombinational events hadtaken place during the DNA replication process. Cells that were treatedin G i phase and allowed to proceed to mitosis in the presence ofbromodeoxyuridine to density label newly replicated DNA were analyzedwith cesium chloride density sedimentation. Unreplicated DNA waspresent in the mitotic cells of the treated populations but not in theuntreated control cells. Further, flow cytometric measurements, madeunder hypotonie conditions in order to reduce chromatin condensationeffects, demonstrated that the mitotic cells from the mitomycin C-treatedpopulations contained 10-20% less DNA than untreated mitotic controls.These results indicate that chromosome breaks induced by mitomycin Care the result of cells reaching mitosis without having fully completedDNA replication.

INTRODUCTION

Many chemical and physical agents used in the treatment ofmalignancy are known to produce DNA lesions that ultimatelyresult in chromosome aberrations (1). While the existence ofchromosome aberrations has been recognized since Muller's

first description in 1927 (2), little is known about how specifictypes of DNA lesions are translated into aberrations at thechromosomal level (for reviews, see Refs. 3 to 5). The purposeof the studies reported here was to gain a better understandingof how DNA damage induced by MMC4 might cause the

formation of one type of chromosome aberration, e.g., thechromatid break.

Mitomycin C, a therapeutic drug used in the treatment of

Received 2/24/86; revised 4/21/86; accepted 4/22/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grants CA-27931 and CA-39534 from the National

Cancer Institute, NIH.1 Present address: Colorado State University, Department of Radiology and

Radiation Biology. Fort Collins, CO 80523, and the Eleanor Roosevelt Institutefor Cancer Research, 4200 E. 9th Ave.. Denver, CO 80262.

3To whom requests for reprints should be addressed.4The abbreviations used are: MMC, mitomycin C; BrdUrd, bromodeoxyuri

dine: dThd, thymidine; HH, heavy-heavy; HL, heavy-light; LL, light-light; PCC,prematurely condensed chromosomes.

malignancy, covalently binds to the DNA of cells and producesboth monoadducts and DNA-DNA and DNA-protein crosslinks, most likely involving the O6 position of guanine (6).MMC-induced damage results in a reduction in the rate ofDNA replication and a dose-dependent delay in cell cycleprogression (7, 8). At the chromosome level, MMC is thoughtto produce chromosome aberrations by means of an S-phasedependent mechanism; i.e., chromosome aberrations are notobserved unless the treated cell has undergone replicative DNAsynthesis following the induction of DNA damage (9). Whenthe treated cells eventually reach mitosis, chromatid-type breaksand exchanges are observed (10).

The chromatid breaks after MMC treatment are noteworthyin that the deleted regions can be quite lengthy in appearance,suggesting that a portion of the genome might be missing.Similarly, highly damaged metaphase spreads closely resembleS-phase cells which have been induced into premature chromosome condensation. These observations led to the hypothesisthat cells containing MMC-induced damage can eventuallyattain mitosis without having fully replicated their DNA. Somechromosome breaks would therefore be the result of unrepli-cated chromosome regions.

If this hypothesis holds, the cells that reach mitosis afterMMC treatment should exhibit several distinct characteristics.(a) The damaged mitotic cells should still contain MMC-induced DNA lesions, (b) These cells should contain a portionof their DNA in an unreplicated form, (c) The treated cellsshould exhibit a diminished DNA content upon reaching mitosis. In this paper we have tested and confirmed these predictions using a variety of techniques including DNA alkalineelution (to detect residual DNA-DNA and DNA-protein crosslinks), CsCl density sedimentation (to detect unreplicatedDNA), and flow cytometry (to detect mitotic cells with diminished DNA content). Thus, it is likely that MMC-inducedchromatid breaks are a consequence of incomplete DNA replication caused by the presence of residual MMC-induced DNAlesions.

MATERIALS AND METHODS

Cell Culture, Cell Synchronization, and Drug Dilution. HeLa cellswere grown as monolayer cultures on Lux 150-mm dishes in Hsu'smodified McCoy's Medium 5A (GIBCO, Grand Island, NY) supple

mented with 10% fetal calf serum (K C Biological, Lenexa, KS), 3 mML-glutamine (GIBCO), penicillin (100 ^g/ml) (GIBCO), streptomycin(100 ^g/ml) (GIBCO), and 0.1 mM CaCl2 (MCB, Norwood, OH).Cultures were maintained at 37°Cin a humidified 5% CO2 incubator.

The cells were subcultured with 0.125% trypsin (GIBCO). The procedures for obtaining HeLa mitotic cells were described previously (11).Briefly, exponentially growing cells were treated with 2.5 mM dThd for22-24 h, released from the dThd block, and then accumulated in mitosisin the presence of 90 psi nitrous oxide. After selective detachment ofmitotic cells, this procedure routinely yielded populations with a mitoticindex of at least 95%. Upon release from nitrous oxide (N2O), the cellscompleted mitosis and entered <., phase in a synchronous wave.

MMC (Mutamycin, Bristol Laboratories, Syracuse, NY) was dissolved just before use in sterile distilled water to a 100-¿ig/mlstocksolution and then diluted in McCoy's Medium 5A with 1% fetal calf

serum to a final concentration of 14032

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

Experimental Protocol. The general experimental protocol was identical for every type of study described in this paper. HeLa cells weresynchronized to mitosis and selectively detached, and 4 x 106cells were

plated per 150-mm Petri dish. The cells were allowed to divide, attachto the dishes, and progress into GI phase. Five h after plating, the Gìcells (labeling index, <1%) were treated with MMC (1 jig/ml) for 1 h,rinsed 3 times with medium, and reincubated in fresh McCoy's medium

with 10% fetal calf serum. After 18 h, the old medium containingfloating cells was removed, and fresh medium was added. Cells werethen accumulated in mitosis in the presence of N2O, and mitotic cellswere selectively detached for analysis after appropriate time intervals.

For the alkaline elution and CsCI analyses, the cells were radioac-tively labeled with [I4C]dThd (0.02 MCi/ml) (40-60 Ci/mivi) for two full

cell cycles prior to the first synchronization. For the studies concerningdamage to newly replicated DNA, [MC]dThd was added to the cultures

after MMC treatment. The procedure for alkaline elution has beenpreviously described (12). For the CsCI assays of unreplicated DNA,BrdUrd (20 Mg/ml) was added after MMC treatment.

Neutral Cesium Chloride Density Centrifugation. After drug treatmentand cell cycle progression, mitotic cells were accumulated in N2O,selectively detached, centrifuged, and resuspended in Tris buffer (0.15M NaCl:0.01 M EDTA:0.01 M Tris, pH 8.1) to a concentration of IO6/

ml. Cells were lysed by addition of Sarkosyl to 0.1 %, incubated for 30min at 60°Cwith RNase A (80 jig/ml) (Millipore Corporation, Freehold, NJ), and then incubated for 1 h at 60°Cwith Pronase CB (0.5

mg/ml) (Calbiochem-Behring Company). The cell lysate was mixedwith an equal volume of Sevag [chloroform:isoamyl alcohol (24:1, v/v)] at 4°C.After centrifugation, the aqueous phase was removed, and

the Sevag procedure was repeated until the interface between the sampleand Sevag was clear. The DNA was then precipitated with 2 volumesof 95% ethanol at 0°Cand collected by a 20-min centrifugation at 4°C

and 10,000 rpm. The DNA was resuspended in 0.15 M NaCl:0.015 MNa3C6H5O7 (1:100) and used in the preparation of the neutral CsCIdensity gradients as previously described (13). Individual gradient fractions were mixed with 1 ml of water and 10 ml of Scintiverse II (FisherScientific, Houston, TX), and radioactivity was measured with a Packard Model 2650 liquid scintillation spectrometer.

Flow Cytometry Analysis. Samples of mitotic cells designated forflow cytometric DNA content analysis were washed in physiologicalsaline and then exposed to either hypotonie 0.075 M KCl or physiological saline for 25 min in the presence of Colcemid (0.05 Mg/ml).Following centrifugation (3 min at 150 g), the cell pellets were resuspended by mixing on a vortex, fixed in suspension with 70% ethanol(4°C),and stored at 4°Cuntil analysis.

For DNA fluorochrome staining, 2 x IO6cells were resuspended inDulbecco's phosphate-buffered saline containing 1 mM MgCl2 and

centrifuged, 1 ml of 0.5% pepsin (Accurate Chemicals, Hicksville, NY)was added, and the solution was held for 4 min at room tremperature.Digestion was stopped by the addition of 2.5 ml of cold Tris buffer (0.1M Tris base:0.1 M NaCl:0.84 M HCl, pH 7.4). After centrifugation thecell suspension was resuspended in 1.5 ml of ethidium bromide (25 #<g/ml) (Sigma Chemical Co., St. Louis, MO) for IO min, followed by theaddition of 1.5 ml of mithramycin (50 ^g/ml) (Worthington Biochemical Corporation, Freehold, NJ) containing 30 mM MgCl2. Immediatelybefore analysis each sample received 3 drops of 1% RNase (Worthington) and was filtered through a 65-Mm nylon mesh. The integral DNAfluorescence emission per cell was measured with an ICP-11 Phyweinstrument interfaced to a Nuclear Data 256 channel pulse heightanalyzer equipped with an HP-26 48A terminal and tape storage device(14, 15).

Cell Fusion. The procedure for cell fusion has previously been described in detail (16). Briefly, mitotic HeLa cells (mitotic index >95%)were fused with an equal number of MMC-treated HeLa cells usingUV, inactivated Sendai virus. After fusion, the cells were exposed tohypotonie, fixed in methanol:glacial acetic acid (3:1, v/v) dropped onclean, wet slides, and stained with Giemsa.

RESULTS

Visualization of Chromosome Damage after MMC Treatment.When HeLa cells were synchronized in G, phase, treated for 1

h with MMC, and then allowed to progress to mitosis, chro-matid breaks, gaps, and exchanges were observed in metaphasepreparations that were not seen in the untreated controls (Fig.I, A and B). Ninety % of the total aberrations at this dose werechromatid breaks, many of which appeared to be the result ofunusually long chromatid deletions, as well as displaced breaks.

At this high dose of MMC (1 ^g/rnl), many cells in thetreated population are not capable of achieving mitosis andthus remain in interphase for many hours. To visualize thechromosomes in these drug-delayed interphase populations,cells were treated in G, phase for 1 h with MMC, reincubatedin drug-free medium for 70 h, and then fused with mitotic cellsto induce premature chromosome condensation. In controlpopulations, G, cells yield PCC with a single chromatid perchromosome, while Ga cells yield PCC with two chromatidsper chromosome. The PCC of S phase appear pulverized.Nevertheless, both single (unreplicated) and double (replicated)chromosome segments can be observed, with those portions ofthe genome actively undergoing replication appearing diffuse(Fig. 1C) (17, 18). The PCC of the MMC-treated cells resembled "normal" S-phase PCC in the sense that both single and

double segments were present. However, the morphology ofthese PCC was distinct in that little diffuseness was present,and condensed single chromatid elements were directly joinedto condensed double chromatid regions (Fig. 1D). Pulse labeling studies indicated that DNA synthesis was dramaticallyreduced in these cells (data not shown).

The fact that the chromosome preparations of the mitoticcells and the PCC were of similar morphology after MMCtreatment raised the possibility that residual MMC-inducedlesions in the DNA blocked the completion of DNA synthesis,and yet the cells continued to prepare for mitosis and eventuallycondensed their chromatin and entered mitosis. For this possibility to hold, one would expect to find residual MMC-inducedlesions in the mitotic populations of treated cells as well asindications of incomplete DNA replication. The subsequentexperiments were designed to test these expectations.

Residual DNA Damage in MMC-treated Cells at Mitosis. TheDNA alkaline elution technique (19) was utilized to measurethe amount of DNA damage in the HeLa cells after MMCtreatment. With this technique, radioactively labeled cells arelysed directly onto filters, and DNA is eluted from the filter inthe presence of an alkaline solution, pH 12.1. Under theseconditions, the DNA strands unwind and pass through the filterin the order of increasing strand length. The presence of singleor double strand breaks or alkaline labile sites causes moreDNA to elute. On the other hand, DNA-DNA or DNA-proteincross-linking (making DNA strands effectively larger) masksstrand breakage and retards elution.

To determine the degree of MMC-induced DNA damagepresent immediately after treatment, prelabeled G, HeLa cellswere treated with MMC (l ßg/ml)for 1 h and then immediatelyanalyzed by DNA alkaline elution. As shown in Fig. 2, 90 and92.5% of the radioactivity were retained on the filter afterelution for the control and MMC-treated populations, respectively. Thus few strand breaks or alkaline labile sites wereapparent immediately after treatment. However, irradiation ofthe samples on ice just prior to elution demonstrated a decreased rate of elution in the MMC-treated cells compared tocontrols. This suggests that DNA-DNA and DNA-proteincross-links were present in the MMC-treated cells. To distinguish between DNA-DNA and DNA-protein cross-links, thelysed cells were treated with proteinase K prior to elution torelease the DNA-protein cross-links. The fact that proteinaseK treatment resulted in only a slight increase in DNA elution

4033

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN INDUCED CHROMATID BREAKS

4 ^ ^>** » n *T,

-.• .' »;..'•-^.'-••C*'v -••''-. /. '•

V -• >• N"

.•-- ••Fig. I. Chromosome morphology of mitomycin C-treated cells. Synchronized HeLa cells were treated in G, phase with or without MMC for 1 h, washed free of

drug, and reincubated in drug-free medium to allow cell cycle progression. A, mitotic figure of untreated control; fi, mitotic figure of MMC-treated cell demonstratingchromosome damage with many apparent large chromatid deletions (arrows); C, S-phase PCC of an untreated control cell; D, S-phase PCC of MMC-treated cellblocked in S phase demonstrating continuity of single and double chromatid regions (arrows), x ISOO.

(7 and 10% for the control and MMC treatment, respectively)suggests that most of the cross-links induced by MMC areDNA-DNA rather than DNA-protein cross-links.

To determine whether cells treated in d phase could entermitosis despite residual DNA damage, prelabeled HeLa cellswere treated in G, phase with MMC and allowed to progressto mitosis, and then mitotic cells were selectively detached andanalyzed by alkaline elution. As shown in Fig. 3-4, the MMC-treated cells exhibited a slight decrease in DNA retentioncompared to the controls (O versus •,respectively). This suggested the presence of DNA strand breaks or alkaline-labilesites in the MMC-treated cells. When the two populations wereirradiated on ice just prior to elution to allow the detection ofresidual cross-links, the parental DNA of the MMC-treatedcells still exhibited increased DNA retention on the filterscompared to the controls, suggesting the presence of residualcross-links in the MMC-treated cells that attained mitosis (Fig.3A, A versus A, respectively). Proteinase K digestion prior toelution indicated that the cross-links remaining in the treatedcells at mitosis were DNA-DNA interstrand type.

It is not understood how the treated cell might replicate pastcross-linked DNA, but it is feasible that the DNA synthesizedby the cells after MMC might contain breaks in the regions of

residual cross-links in the parental DNA. To test this, theparental DNA was prelabeled prior to MMC treatment in GÃŒphase with [14C]dThd, and then the cells were incubated in[3H]dThd during the S phase after MMC treatment to label the

newly replicated DNA. As before, cells that accumulated atmitosis were selectively detached and analyzed by DNA alkalineelution. Surprisingly, the elution rate of 3H-labeled DNA syn

thesized after the MMC treatment was equally retarded as thatof the parental DNA (Fig. 3B). This suggests that DNA synthesized after MMC treatment also contained DNA-DNAcross-links.

That DNA-DNA interstrand cross-links that were present inequivalent amounts in the parental and newly replicated DNAafter MMC treatment might be a consequence of one of threemechanisms, (a) The conditions used for liquid scintillationcounting or double radioactive labeling might have producedan artifact, (b) The formation of MMC-induced cross-linksmight occur by a two-step mechanism over a period of hourssimilar to that described for ci's-platinum (20), nitrosoureas

(21), and melphalan (22). (c) A recombinational mechanismmight be involved during replication past cross-links (see "Discussion").

The first mechanism was tested by not prelabeling the paren-

4034

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

tal DNA but labeling only the newly replicated DNA afterMMC treatment. The elution profiles of these cells after pro-teinase K digestion were very similar to those seen in the doublelabel experiments (data not shown). This result indicates thatthe double radioisotope labeling procedure was not interferingwith the assay.

The second possible explanation involves a time-dependentmechanism for cross-link production. If interstrand cross-linkswere formed in two discrete steps over a period of time, the

1 00 _

zLU

zQ

LU

OocLU

FRACTION NUMBERFig. 2. DNA alkaline elution analysis of control and treated Gt HeLa cells

immediately after MMC treatment. O, untreated control; •.MMC-treated population: A, MMC-treated cells irradiated on ice just prior to elution to reveal thepresence of DNA cross-links: Q, MMC-treated cells irradiated on ice and treatedwith proteinase K prior to elution to release DNA-protein cross-links; A. untreatedcontrol irradiated on ice just prior to elution: •.untreated control irradiated onice and treated with proteinase K just prior to elution.

maximum cross-link levels produced would not be observedimmediately after MMC treatment but at some later time (i.e.,6-17 h after drug treatment). Thus if the first step of cross-linkformation occurred in G, phase while the second step occurredafter some DNA replication had occurred, it would be possiblefor newly replicated DNA to be involved in cross-links. Todetermine whether MMC forms cross-links by a similar mechanism, the parental DNA was prelabeled with [l4C]dThd, and

the cells were treated with MMC as before in G, phase. Treatedand control populations were harvested for alkaline elutionanalysis at 0, 6, and 15 h after MMC treatment. To allowdetection of cross-links, the cells were irradiated on ice with400 rads of 7-rays just prior to elution. As shown in Fig. 44,fewer cross-links were present in the MMC-treated cells 6 hafter treatment compared to that observed immediately aftertreatment as evidenced by a decreased DNA retention (D versusA, respectively). The decrease in retention for the MMC-treatedcells was not due to the initiation of DNA synthesis becauseincreased elution kinetics was not observed in the unirradiatedcontrol samples (data not shown), and the number of nicksproduced by the irradiation is large compared to the number ofnicks associated with DNA replication. Thus the drop in filterretention reflected repair of DNA-DNA cross-links. Similarly,the degree of cross-linking evident in the parental DNA wasalso decreased at 15 h posttreatment when compared to thatobserved immediately after treatment (Fig. 4Ä).Since the maximum cross-link levels were observed immediately after MMCtreatment, these results suggest that a delayed two-step mechanism for cross-linked formation could not explain the findingthat cross-links become associated with DNA synthesized afterMMC treatment.

Demonstration of Unreplicated DNA in Mitotic Cells afterMMC Treatment. The cytological observations described earliersuggested that some MMC-treated cells might enter mitosiswithout completing DNA replication. If so, the cells reachingmitosis would be expected to contain some unreplicated DNA.This was tested by prelabeling cells with [3H]dThd for two cell

generations, synchronizing to GÃŒphase, and treating withMMC as before. After drug treatment, the cells were reincu-bated in medium containing BrdUrd to density-label the newlyreplicated DNA. After appropriate periods of time, the cellsattaining mitosis were accumulated in N2O, selectively detached(mitotic index, >95%), and the DNA was isolated and subjectedto neutral CsCl density gradient analysis. In this assay, BrdUrd-

Fig. 3. DNA alkaline elution analysis ofcells treated in G, with or without MMC andselectively detached at mitosis. A, parentalDNA labeled prior to MMC treatment: B.daughter DNA labeled after MMC treatmentand prior to selection of cells at mitosis. •,untreated control O, MMC-treated cells; A,MMC-trealed cells irradiated on ice just priorto DNA elution: A, untreated control cellsirradiated on ice prior to DNA elution.

80 _ Z 80O

246

FRACTION NUMBER

468

FRACTION NUMBER

4035

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Fig. 4. Alkaline elution analysis of cell populations al various times after MMC treatmentin Gìphase compared to that found immediately after MMC treatment. A, 0 and 6 h post-MMC treatment; B, 0 and 15 h post-MMCtreatment. •.untreated controls immediatelyafter sham MMC treatment: O, untreated controls at 6 or 15 h after sham treatment; A.MMC-treated cells immediately after treatment; D, MMC-lreated cells at 6 or 15 h aftertreatment. All populations were irradiated onice just prior to DNA elution.

MITOMYCIN-INDUCED CHROMATID BREAKS

1 1 1 1 1 1 T100

2 4 6 I

FRACTION NUMBER

20 _

2461

FRACTION NUMBER

containing replicated DNA can be separated from unreplicatedDNA on the basis of density.

As shown in Fig. 5A, the untreated control mitotic preparations yielded one major DNA peak present at a density of 1.75g/cm\ typical of DNA replicated once in the presence ofBrdUrd. By weight, the LL region of the control comprised3.8% of the gradient compared to 85.8% in the HL region. Incontrast, DNA from the MMC-treated populations selected atmitosis contained two distinct peaks, with 18.5% of the gradientin the LL peak (1.71 g/cm3) and 67.8% in the HL peak (1.75g/cm1) (Fig. 5B). In a repeat experiment, approximately 15%of the gradient of the MMC-treated sample was found in theLL region, typical of unreplicated DNA. Since the mitoticindices of the analyzed populations were all greater than 95%,these results suggest that the MMC-treated populations arereaching mitosis with some unreplicated DNA.

Detection of Decreased DNA Content in Mitotic Cells afterMMC Treatment. The preceding experiments showed that cellsthat enter mitosis after MMC treatment still contain detectableDNA-DNA interstrand cross-links and some unreplicatedDNA. If chromosome breaks were a consequence of the cell's

inability to complete DNA replication past the residual DNAdamage, one would also expect these drug-treated cells to havea deficiency in DNA content at mitosis. To test this, the DNAcontents of control and MMC-treated populations selected atmitosis were determined by flow cytometry using ethidiumbromide and mithramycin as the fluorescent dyes (23, 24).

Since some drugs have been shown to interfere with mithramycin staining (25), and since the presence of interstrand crosslinks could conceivably diminish the binding of the intercalatingdye ethidium bromide, it was first necessary to ensure thatMMC treatment alone would not affect the DNA contentmeasurements. To test this, HeLa mitotic cells were treatedwith up to 100 fig of MMC per ml for l h in the presence ofColcemid, and then the cells were analyzed for DNA content.No difference in the measured DNA content due to MMCtreatment was observed (data not shown). Similar results werealso obtained in MMC-treated mitotic cells exposed to a hypotonie solution (0.075 M KC1) for 25 min prior to fixation.Thus, the presence of even high levels of DNA-DNA crosslinks does not interfere with the accurate determination of DNAcontent.

ü A.>-• •

r 3

oato

T3çaŒ 0

10 20 30

Fraction Number40

20 30

Fraction Number40

Fig. 5. Cesium chloride analysis of mitotic populations of cells treated in G,with or without MMC. washed free of drug, reincubated in medium containingBrdUrd to density label newly replicated daughter DNA, and selectively detachedat mitosis. A, untreated controls: B, MMC-treated population. Note the presenceof a significant light-light peak in the MMC-treated population demonstratingthe presence of unreplicated and/or recombined DNA.

4036

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

Fig. 6. Flow cytophotometrie analysis ofmitotic HeLa cell populations treated in dwith or without MMC, allowed to progress tomitosis, and selectively detached. I. untreatedcontrols and MMC-treated populations without hypotonie pretreatment of populations; B,untreated controls and MMC-treated populations with hypotonie pretreatment to eliminatedye-uptake artifacts due to differences in chro-matin structure. Internal control lymphocytepopulations were placed at channel 17, and 5-10 X IO3cells were counted for each measure

ment.

10

8

6

o

010

= 80)O c

-AControl

MMC

ILAA ! ! I ! .!. I. I I I I I I I I

o

EsZ

0)

U

I I I I I I Iio|-B

8

6

4

2

O10

8

6

16 32 48 64 80 96 112Channel Number

I I I I I I I

Control

MMC

16 32 48 64 80 96 112Channel Number

To determine whether cells treated in G¡could come tomitosis with a deficiency in DNA content, HeLa cells weretreated in G! with MMC, selectively detached at mitosis asdescribed above. The cells were then analyzed by flow cytometryusing human peripheral blood lymphocytes as an internal DNAcontent marker. The DNA content measurements for the control (mitotic index, 95%) and MMC-treated (mitotic index,96%) cell populations are shown in Fig. 6A. While it wasexpected that the drug-treated cells would exhibit less fluorescence than the controls due to a diminished DNA content, justthe opposite was observed (modal peak channel numbers of 52and 50 for the MMC-treated and control populations, respec

tively).These results suggested that either the MMC-treated cells

reached mitosis with an increased DNA content or that thechromatin of MMC-treated cells was more open, allowingincreased dye binding. The latter notion is possible because ithas previously been shown that mitotic cells blocked withColcemid for prolonged time periods have their peak channelsdiminished by as much as 20% (26). Also, PCC studies haveshown that MMC treatment induces chromatin elongationduring the repair process (27). To minimize the effects ofchromatin condensation differences on DNA content measurements, the MMC-treated and control populations were exposedto hypotonie solution (0.075 M KC1) prior to fixation andstaining with mithramycin and ethidium bromide. Preliminarystudies showed that the greatest dye binding was observed incells swollen with hypotonie solution for 25 min (data notshown). As shown in Fig. 6Ä, when the MMC-treated andcontrol populations were analyzed by flow cytometry afterhypotonie exposure, the control cells now showed increasedDNA-bound dye when compared to the MMC-treated cells(modal peak channel numbers of 57 and 52, respectively). Thusthe MMC-treated cells appear to reach mitosis with a decreasedDNA content.

Interestingly, with hypotonie treatment, the control population increased in peak channel number from 50 to 57 (a net14% increase), while the MMC-treated cells remained at channel 52. The result with the control population is consistent withthe observation of others that hypotonie treatment increases

dye binding (28, 29). The contrasting result with the MMC-treated cells suggests that the chromatin of these cells is alreadyin a conformation that allows maximum dye binding. Assuminga HeLa cell in d phase contains 17 pg of DNA while a normalhuman lymphocyte contains 6.5 pg of DNA, the MMC-treatedcells appear to be lacking 9-10% of their expected DNA whenthey reach mitosis. In a repeat experiment, a 20% difference inDNA content was observed between the MMC and controlmitotic populations. These figures are on the same order ofmagnitude as those obtained by cesium chloride measurementsof the amount of unreplicated DNA in the MMC-treated populations.

DISCUSSION

The purpose of this study was to determine how MMC-induced DNA lesions present during S phase induce chromosome aberrations. The preceding experiments demonstrate thatMMC-treated cells reach mitosis without completing eitherDNA damage repair or DNA replication and with a decreasedDNA content. This suggests that chromosome breaks resultfrom incomplete DNA replication prior to mitosis. A similaridea has been previously postulated to explain the formation ofbreaks after fluorodeoxyuridine treatment (30).

DNA alkaline elution analysis of the cells reaching mitosisdemonstrated measurable residual DNA-DNA cross-links inthe mitotic populations of cells that had been treated in d withMMC. The fact that cross-links can persist in cells for extendedperiods of time is consistent with results from several otherstudies. Palitti et al. (31) showed that some MMC-induceddamage remains in Chinese hamster ovary cells until 62 phase,since their repair could be inhibited with hydroxyurea, aphidi-colin, or caffeine. Murnane and Byfield (32) reported that aportion of the cross-links induced in C3H10T'/2 cells by nitro

gen mustard, phosphoramide mustard, or melphalan was notrepairable even though the cells were cross-link repair proficientif challenged with a second drug dose. Similarly, Sognier andHittelman (33) reported that, although some initial repair wasobserved during the first 48 h after MMC treatment of density-inhibited normal human fibroblasts, measurable cross-linkingremained in the cells for at least 96 h after a 1-h treatment with

4037

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

G, PHASE S PHASE

I

Fig. 7. Diagrammatic representation of a postulated mechanism for MMC-induced chromatid break formation involving inability to complete DNA replication past cross-links and recombinational events occurring at the sites of blocksto replication.

MMC (3 ¿¿g/ml).Slow repair kinetics of MMC-induced crosslinks was also reported by Dorr et al. (34). These studies allsuggest that a fraction on the cross-links induced in cellularDNA might be irrepairable. Indeed, some evidence indicatesthat there might be regions of the genome that are resistant torepair functions (35).

Since DNA damage in these studies was measured withalkaline elution, only the presence or absence of breaks andcross-links was measured. However, MMC induces monoad-ducts at 5 times the frequency of cross-links (36). It is thereforenot possible to exclude a contributory role for monoadducts onthe effects observed. However, it is unlikely that monoadductsare primarily responsible for these results for several reasons.(a) Monofunctional alkylating agents are less efficient inducersof chromsosome aberrations than polyfunctional alkylatingagents (37); (b) O6-alkyl guanine adducts are poor inducers of

chromosome aberrations, and some cell lines can successfullyreplicate past these lesions (38, 39); (c) the repair rates for O6-

alkyl guanine are relatively short in human cells due to thepresence of alkyltransferase, so many of the lesions would berepaired before DNA replication was initiated (40); and (d)damage induced by bifunctional MMC is potentiated by hy-droxyurea in G? while damage induced by monofunctionalMMC is not (31).

It is not known how cells replicate past lesions such as intactDNA-DNA cross-links. Cleaver has suggested that the presenceof two inhibitory DNA lesions in a replicón might preventsynthesis of the DNA between the lesions (41). Interestingly,however, our DNA alkaline elution data suggest that the DNA-DNA cross-links become associated with DNA synthesizedafter drug treatment. Since control experiments showed thatthis result was not a consequence of a delayed two-step mechanism of cross-link formation nor an artifact of the radioisotopelabeling and counting conditions, this finding suggests thatsome form of recombinogenic process takes place when the cellreplicates past these DNA lesions. One possible mechanism forthis process is illustrated in Fig. 7 where two cross-links withina replication unit prevent the completion of DNA synthesisbetween the cross-links. If a recombinogenic process occurredat or near the cross-links, the resultant structure would satisfyour observations; i.e. (a) cross-links would be present and wouldbe distributed in both parental and daughter DNA; (b) bothparental and daughter DNA would also have single strandbreaks; and (c) a portion of the genome would remain unrepli-

cated.A number of investigators have attempted to demonstrate

the existence of recombination in mammalian cells by lookingfor the presence of HH DNA in CsCl density gradients. However, only very small amounts of HH DNA (i.e., 0.1-0.2%)

MITOSIS were ever detected in these reported studies (42, 43), and it wasbelieved that recombination probably did not occur in mammalian cells. On the other hand, recent evidence has suggestedthat recombination can occur in mammalian cells after damaging treatment. Using UV endonuclease to probe the locationof thymidine dimers in 3T3 cellular DNA after UV treatment,Meneghini et al. (44) showed that dimers originally inducedinto parental DNA were transferred to newly synthesized DNA.Earlier studies had failed to show this effect, since it mainlyoccurs soon after damage induction and declines thereafter.Subsequently, Fournace (45) confirmed Meneghini's results

using DNA alkaline elution. Interestingly, a mammalian enzyme similar to the RecA protein in E. coli, known to beinvolved in recombinational events, has recently been isolated(46).

We have shown in this paper that cells reaching mitosis aftertreatment in GI phase with MMC contain unreplicated DNAand exhibit a decreased DNA content when compared to untreated control populations. In previous reports, other investigators reached a different conclusion when they measured DNAcontent after either UV irradiation and caffeine (47) or X-raytreatment (48). Although these studies involved different damaging agents, it is possible that diminished DNA content wasactually present in the treated populations but was not detectable using standard flow cytophotometry methods. The resultspresented here suggest that an altered chromatin configurationmight be present in the damaged cells that allows increasedfluorescent dye uptake compared to the control cells. Indeed, ithas recently been demonstrated that agents such as UV irradiation and alkylating agents induce a chromatin decondensationprocess that can be visualized in the interphase PCC (27, 49-51) as well as in mitotic figures (52). However, hypotonietreatment of the cell populations prior to flow cytophotometricanalysis decreases the influence of chromatin condensationdifferences between the two populations and allows a moreaccurate estimation of DNA content.

The results of this study raise the question of how a cell canproceed to mitosis with unrepaired and unreplicated DNA. Thetransition to mitosis in treated cells can be thought of as aproduct of two counterbalancing processes. First, as the cellproceeds through the cell cycle, chromosome condensation/mitotic factors gradually accumulate to prepare the cell formitosis (53, 54). On the other hand, cell fusion studies haveshown that both S-phase cells and damaged quiescent cells canblock G2 cells from progressing to mitosis (55), and extractsfrom damaged cells have been shown to contain factors thatbind to and inactivate mitotic factor activity (56, 57). Thus,when a cell has unrepaired or extensive damage, it often becomes blocked in S or G2 phase, and only the least damagedcells reach mitosis (58, 59). Eventually, however, the decondensation activity may subside, and the accumulated mitotic condensation factors finally force the cell to enter mitosis eventhough neither DNA repair nor DNA replication is complete.A similar situation has been observed in cells that exhibit atemperature-sensitive mutation for S-phase functions (60).After periods of time at nonpermissive temperature, these cellsstill undergo chromosome condensation without completion ofDNA synthesis and exhibit an S-phase PCC morphology. Further, these cells can induce PCC in other cells, suggesting thateven though they cannot complete DNA replication, they stillcontinue to produce chromosome condensation factors (61).Taken together, the results of this study suggest the followingmodel for chromosome break production. Due to the presenceof residual DNA lesions, the treated cells are unable to completeDNA replication. Nevertheless, some cells continue to preparefor and eventually enter mitosis. The unreplicated portion of

4038

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

the chromosome would then appear as a chromatid deletion atmitosis.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Todd Johnson for help in the flowcytophotometric measurements; Josephine Neicheril for assistance inpreparation of the manuscript; James L. Cooper for computer programming and use of equipment; and Dr. Ramesh C. Adlakha, Dr. FrancesM. Davis, and Dr. Potu N. Rao for encouragement and helpful discussions.

REFERENCES

1. Kihlman, B. A. Actions of Chemicals on Dividing Cells. Englewood Cliffs,NJ: Prentice-Hall, 1966.

2. Muller, H. J. Artificial transmutation of the gene. Science (Wash. DC), 66:84-87, 1927.

3. Bender, M. A. Relationship of DNA lesions and their repair to chromosomalaberration production. Basic Life Sci., 15: 245-265, 1980.

4. Evans, H. J. Molecular mechanisms in the induction of chromosome aberrations. In: D. Scott, B. A. Bridges, and F. H. Sobéis(eds.). Progress inGenetic Toxicology, pp. 57-74. Amsterdam: North-HollandrElsevierBiomedicai Press, 1977.

5. Kihlman, B. A. Molecular mechanisms of chromosome breakage and rejoining. Adv. Cell Mol. Biol., /: 59-107, 1971.

6. Tomasz, M., Mercado, C. M., Olson, J., and Chatterjee, N. The mode ofinteraction of mitomycin C with deoxyribonucleic acid and other polypeptidesin vitro. Biochemistry, 13: 4878-4887, 1974.

7. Barlogie, B., and Drewinko, B. Lethal and cytokinetic effects of mitomycinC on cultured human colon cancer cells. Cancer Res.. 40: 1973-1980, 1980.

8. Shatkin. A. J.. Reich. E., Franklin. R. M., and Tatum, E. L. Effect ofmitomycin C on mammalian cells in culture. Biochim. Biophys. Acta, 55:277-289, 1962.

9. Crooke, S. T. Mitomycin C—an overview. In: S. T. Crooke and A. W.Prestayko (eds.). Cancer and Chemotherapy. Vol. 3. pp. 49-60. New York:Academic Press, 1980.

10. Doi, O., Takai, S., Aoki, Y., Higashi. H.. and Kosaki, G. Effects of mitomycinC on HeLa cells at various stages of the division cycle. Gann, 58: 125-137,1967.

11. Rao, P. N. Mitotic synchrony in mammalian cells treated with nitrous oxideat high pressure. Science (Wash. DC), 160: 774-776, 1968.

12. Sognier, M. A., and Hittelman, W. N. The repair of bleomycin induced DNAdamage and its relationship to chromosome aberration repair. Mutât.Res..02:517-527, 1979.

13. Kondoleon, S. K., and Stambrook, P. J. Replication of mammalian DNA inBrdU: appearance of a component with intermediate density. Mol. Gen.Genet.. ISO: 523-530, 1980.

14. Dean, P. N., and Jeff, J. H. Mathematical analysis of DNA distributionderived from flow microfluorometry. J. Cell Biol.. 60: 523-527. 1974.

15. Johnston, D. A., White, R. A., and Barlogie, B. Automatic processing andinterpretation of DNA distributions: comparisons of several techniques.Comp. Biomed. Res., II: 393-404, 1978.

16. Sognier, M. A., and Hittelman, W. N. Effect of DNA repair inhibition oninduction and repair of bleomycin induced chromosome damage. Mutât.Res., 60:61-72, 1979.

17. Johnson, R. T., and Rao, P. N. Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature (Lond.), 226:717-722, 1970.

18. Sperling, K., and Rao, P. N. Mammalian cell fusion. V. Replication behaviorof heterochromatin as observed by premature chromosome condensation.Chromosoma (Beri.), 45: 121-131, 1974.

19. Kohn, K. W., Ewig, R. A., Erickson, L. C., and Zwelling, L. A. Measurementof strand breaks and cross-links by alkaline elution. In: E. C. Friedberg andP. C. Hanawalt (eds.), DNA Repair—A Lab Manual of Research Procedures,Part B, pp. 379-401. New York: Marcel Dekker, Inc., 1981.

20. Zwelling, L. A., Kohn, K. W., Ross, W. E., Ewig, R. A. G., and Anderson.T. Kinetics of formation and disappearance of a DNA cross-linking effect inmouse leukemia L1210 cells treated with cis- and fra/is-diamminedichloro-platinum(Il). Cancer Res., 38: 1762-1768, 1978.

21. Ewig, R. A. G., and Kohn, K. W. DNA damage and repair in mouse leukemiaL1210cells treated with nitrogen mustard, l,3-bis(2-chloroethyl)-l-nitrosou-rea, and other nitrosoureas. Cancer Res., 37: 2114-2122, 1977.

22. Ross, W. E., Ewig, R. A. G., and Kohn, K. W. Differences between melphalanand nitrogen mustard in the formation and removal of DNA cross-links.Cancer Res., 38: 1502-1506, 1978.

23. Crissman, H. A., Mullaney, P. F., and Steinkamp, J. A., Methods andapplications of flow systems for analysis and sorting of mammalian cells.Methods Cell Biol., 9: 179-246, 1975.

24. Kerker, M., Van Dilla, M. A., Brunsting, A., Kratohuil, J. P.. Hsu, P., Wang,D. S., Gray, J. W., and Langlois, R. G. Is the central dogma of flow cytometry(i.e., fluorescence intensity is proportional to cellular dye content) true?Cytometry. 5:71-78, 1982.

25. Alabaster, O., Tannenbaum, E., Habbersett, M. C., McGrath. I., and Herman, C. Drug induced changes in DNA fluorescence intensity detected byflow microfluorometry and their implications for analysis of DNA contentdistributions. Cancer Res., 38: 1032-1035, 1978.

26. Haag, D., Unlweauxhunfwn zur Proportionalität zwischen DNA-Gehalt undfluoreszenzintensitat fluorochromierter ein zelzellen. Histochimie, 36: 283-291, 1973.

27. Hittelman, W. N. Prematurely condensed chromosomes: a model system forvisualizing effects of DNA damage, repair, and inhibition at the level ofchromosome structure. In: A. Collins, S. Downes, and R. T. Johnson (eds.),DNA Repair and Its Inhibition, pp. 341-371. Oxford: IRL Press, 1984.

28. Garcia, A. M. Studies on DNA in leukocytes and related cells of mammals.VI. The Feulgen-DNA content of rabbit leukocytes after hypotonie treatment.J. Histochem. Cytochem., 17:47-55, 1969.

29. Ringertz, N. R., Gledhill, B. L., and Darzynkiewicz, Z. Changes in deoxyri-bonucleoprotein during spermiogenesis in the bull. Exp. Cell Res., 62: 204-218, 1970.

30. Taylor, J. H., Haut, W. F., and Tung, J. Effects of fluorodeoxyuridine onDNA replication, chromosome breakage, and reunion. Proc. Nati. Acad. Sci.USA, 48: 190-198, 1962.

31. Palitti, F., Tanzarella, C., Degrassi, F., De Salvia, R., Fiore, M., and Nata-rajan, A. T. Formation of chromatid type aberrations in the G2 stage of thecell cycle. Mutât.Res., 110: 343-350, 1983.

32. Murnane, J. P., and Byfield, J. E. Irrepairable DNA cross-links and mammalian cell lethality with bifunctional alkylating agents. Chem.-Biol. Interact., J«:75-86, IMI.

33. Sognier, M. A., and Hittelman, W. N. Loss of repair of DNA interstrandcrosslinks in Fanconi's anemia cells with culture age. Mutât.Res., 108: 383-

393, 1983.34. Dorr, R. T., Bowden, G. T., Alberts, D. S., and Liddil, J. D. Interactions of

mitomycin C with mammalian DNA detected by alkaline elution. CancerRes., «.-3510-3516, 1985.

35. Wilkins, R. J.. and Hart. R. W. Preferential DNA repair in human cells.Nature (Lond.), 247: 35-36, 1974.

36. Lown, J. W. The molecular mechanism of antitumor action of the mitomy-cins. In: S. K. Carter and S. T. Crooke (eds.), Mitomycin C—Current Statusand New Developments, pp. 5-26, New York: Academic Press, 1979.

37. Sturelid, S. Chromosome breaking capacity of tepa and analogues in Viciafaba and Chinese hamster cells. Hereditas, 68: 265-276, 1971.

38. Abanobi, S. E., Columbano, A., Mulivar, R. A., Rajalakshmi, S., and Sarma,D. S. R. In vivo replication of hepatic deoxyribonucleic acid of rats treatedwith dimethylnitrosamine: presence of dimethylnitrosamine-induced O6-methylguanine, A^-methyl guanine, and W'-methyl adenine in the replicatedhybrid deoxyribonucleic acid. Biochemistry, 19: 1382-1387, 1980.

39. Ledda, G. M., Columbano, A., Rao, P. M., Rajalakshmi, S., and Sarma, D.S. R. In viro replication of carcinogen-modified rat liver DNA: increasedsusceptibility of O'-methylguanine compared to W-methylguanine in replicated DNA to SI nuclease. Biochem. Biophys. Res. Commun., 95:816-821,1980.

40. Medcalf, A. S. C., and Lawley, P. D. Time course of O'-methylguanineremoval from DNA of A'-methyl-jV-nitrosourea treated human fibroblasts.Nature (Lond.), 289: 796-798. 1981.

41. Cleaver, J. E. Regulation of the responses to DNA damage in the hypersen-sitivity diseases and chromosome-breakage syndromes. In: J. German (ed.).Chromosome Mutation and Neoplasia, pp. 235-249. New York: Alan R.Liss, Inc., 1983.

42. Loveday, K. S., and Latt, S. A. Search for DNA interchange correspondingto SCE in CHO cells. Nucleic Acids Res., 5: 4087-4104, 1978.

43. Romelaere, J.. and Miller-Faures. A. Detection by density equilibrium cen-trifugation of recombination like DNA molecules in somatic mammaliancells. J. Mol. Biol., 98: 195-218, 1975.

44. Meneghini, R., Menck, C. F. M., and Schumacher, R. I. Mechanisms oftolerance to DNA lesions in mammalian cells. Q. Rev. Biophys., 14: 381-432, 1981.

45. Fournace, A. J., Jr. Recombination of parental and daughter strand DNAafter UV irradiation in mammalian cells. Nature (Lond.), 304: 532-534,1983.

46. Kenne, K., and Ljungquist, S. A DNA-recombinogenic activity in humancells. Nucleic Acids Res.. 12: 3057-3068, 1984.

47. Cremer, C., Cremer. J., and Gray, J. W. Induction of chromosome damageby UV light and caffeine: correlation of cytogenetic evaluation and flowkaryotype. Cytometry, 2: 287-290, 1982.

48. Griffiths. T. D., and Tolmach, L. J. Age dependence of X-ray induceddeficiency in DNA synthesis in HeLa S3 cells during Generation 1. Radiât.Res., 63: 501-520, 1975.

49. Hittelman, W. N., and Pollard, M. Induction and repair of DNA andchromosome damage by neocarzinostatin in quiescent normal human fibroblasts. Cancer Res., 42: 4584-4590. 1982.

50. Schor, S. L.. Johnson. R. T., and Waldren, C. A. Changes in the organizationof chromosomes during the cell cycle: response to ultraviolet light. J. CellSci., 17: 539-565, 1975.

51. Waldren, C. A., and Johnson. R. T. Analysis of interphase chromosomedamage by means of premature chromosome condensation after X- andultraviolet-irradiation. Proc. Nati. Acad. Sci. USA, 71: 1137-1141, 1974.

52. Mullinger, A. M., and Johnson, R. T. Manipulating chromosome structureand metaphase status with ultraviolet light and repair synthesis inhibitors. J.Cell Sci., 73: 159-186. 1985.

53. Rao. P. N., and Adlakha. R. C. Chromosome condensation and deconden-sation factors in the life cycle of eukaryotic cells. In: A. L. Maizel and R.Ford (eds.). Mediators in Cell Growth and Differentiation, pp. 45-69. NewYork: Raven Press. 1985.

54. Sunkara, P. S., Wright. D. A., and Rao, P. N. Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes. Proc. Nati. Acad. Sci. USA, 76: 2799-2802,1979.

4039

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MITOMYCIN-INDUCED CHROMATID BREAKS

55. Rao, P. N., and Smith, M. L. Differential response of cycling and non- Visualization of X-ray induced chromosome damage in interphase cells,cycling cells to inducers of DNA synthesis and mitosis. J. Cell Biol., 88:649- Mutât.Res., 23: 251-258, 1974.653, 1981. 59. Hittelman, W. N.. and Rao, P. N. Bleomycin induced damage in prematurely

56. Adlakha, R. C.. Sahasrabuddhe, C. G., Wright, D. A., and Rao, P. N. condensed chromosomes and its relationship to cell cycle progression inEvidence of the presence of inhibitors of mitotic factors during G, period in n c"° cells- CanE." Res-<34: ^l33"?43'•' 9D74'

.. n i /•Tin- r n-r n/n mi inoi 60. Nishimoto, T., Eilen, E., and Basilico, C. Premature chromosome conden-mammalian cells^J. Cell¡B.ol.,97: 1707-1713. 1983 sation in a ts DNA~ mutant of BHK cells. Cell, 75: 475-483, 1978.57. Adlakha, R. C.. Wang. Y. C.. Wright, D. A., Sahasrabuddhe, C. G., Bigo, 6, Hayashi A Yamamoto. s.. Nishimoto. T., and Takahashi, T. Chromosome

H., and Rao, P. N. Inactivation of mitotic factors by U. V. irradiation of condensing factor(s) induced in tsBN2 cells at a nonpermissive temperature:HeLa cells in mitosis. J. Cell Sci., 65: 279-295, 1984. evidence for transferable material by cell fusion. Cell Struct. Fund., 7: 291-

58. Hittelman. W. N., and Rao. P. N. Premature chromosome condensation. I. 294, 1982.

4040

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1986;46:4032-4040. Cancer Res   Marguerite A. Sognier and Walter N. Hittelman  Consequence of Incomplete DNA ReplicationMitomycin-induced Chromatid Breaks in HeLa Cells: A

  Updated version

  http://cancerres.aacrjournals.org/content/46/8/4032

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/46/8/4032To request permission to re-use all or part of this article, use this link

on June 18, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from