[CANCER RESEARCH 45, 5321-5327, November 1985]

DifferentialSensitivityof a Mouse MyeloidLeukemiaCell Lineand NormalMouse Bone MarrowCells to X-Ray-inducedChromosomeAberrations1

Marilyn J. Aardema, William W. Au,2 Russell E. Hand, Jr., and R. Julian Preston

University of Tennessee-Oak Ridge Graduate School of Biomédical Sciences [M. J. A.] and Oak Ridge National Laboratory [W. W. A., R. E. H., R. J. P.], Oak

Ridge, Tennessee 37831

ABSTRACT

Cell line ML-1 was established from a myelogenous leukemiaof an RFM mouse. The ML-1 cells and in vitro normal mouse

bone marrow cells were analyzed to determine if there was adifferential sensitivity to X-ray-induced chromosome aberrations

in Gìcells and/or differences in postirradiation cell cycle progression. Cells identified as being in G, at the time of irradiation bytheir staining pattern after replication in 5-bromodeoxyuridine

were analyzed for all types of chromosomal aberrations followingX-ray doses of 0.5, 1.0, 1.5, and 2.0 Gy. ML-1 cells showed agreater sensitivity to the induction of both chromosome-typeaberrations (dicentrics and terminal deletions) and chromatid-

type aberrations (exchanges and deletions) compared to normalmouse bone marrow cells, which only contained chromosome-type aberrations. The presence of chromatid-type aberrations inthe ML-1 cells and not normal bone marrow cells suggested a

differential progression through the cell cycle for the two celltypes after irradiation. Mitotic index and flow cytometric analyseswere performed and showed that both cell types have a delay inprogression from G2 into mitosis, but only the normal mousebone marrow cells have a delay in progression from Gìinto S,as well as delayed progression through the S phase following X-irradiation. These results indicate that the ML-1 leukemia cells

have an increased radiosensitivity. This may be due to a defectin their ability to respond to DNA damage as evidenced by theirlack of a G,- and S-phase delay which allows normal cells an

increased time to repair DNA damage before replication. Thesesame characteristics have been observed in ataxia telangiectasiacells and may well represent a general feature of cells withincreased radiosensitivity.

INTRODUCTION

The relationship between cellular sensitivity to cytotoxicagents, cell cycle delay, and DNA repair has been investigatedusing mammalian cell lines which show a differential sensitivityto the induction of chromosome damage, cell killing, and/ormutation compared to normal cells. Cells derived from inheritedhuman disorders which show an increased sensitivity to variousagents have been examined for possible repair and/or replicationdefects. These include AT3 cells which are sensitive to X-rays

1Research sponsored jointly by NIH Training Grant NIH-GM 7438 in genetics

and the Office of Health and Environmental Research, United States Departmentof Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy

Systems, Inc.2 Present address: Division of Environmental Toxicology, Department of Preven

tive Medicine and Community Health, The University of Texas Medical Branch,Galveston, TX 77550.

3The abbreviations used are: AT, ataxia telangiectasia; FA, Fanconi's anemia;

XP, xeroderma pigmentosum; HN2, nitrogen mustard; Ml, mitotic index; BrdUrd,5-bromodeoxyuridine; NUPD, nonunion proximal and distal; RB, retinoblastoma.

Received 4/29/85; revised 8/7/85; accepted 8/12/85.

(1-4), bleomycin (5), 4-nitroquinoline 1-oxide (6), and neocarzin-ostatin (7); FA cells which are sensitive to cross-linking agents(8,9), bleomycin (9), and X-rays (1); Bloom syndrome cells whichare sensitive to X-rays (1); and XP cells which are sensitive to

UV (10, 11). There are conflicting reports concerning repair orreplication defects in these syndromes, but no specific defectwhich could completely account for the differential sensitivity hasbeen found except for XP cells, which clearly show a deficientrepair of UV-induced pyrimidine dimers (10,12).

Rodent cell lines have also been used to study mechanismsof differential sensitivity. The V79/79 Chinese hamster cell linehas been shown to be more sensitive than the parental V79 lineto X-rays (13), UV, methyl nitrosourea, ethyl methanesulfonate

(14), methyl methanesulfonate, and HN2 (15). Mouse lymphomaL5178Y sublines show an increased sensitivity to X-rays compared to the parent L5178Y line (16-20). The rat Yoshida lym-

phosarcoma cell line has an increased sensitivity to méthylènedimethane sulfonate, UV (21), X rays, HN2, and sulfur mustard16). As with the human cell lines, no specific defect has beenfound in these rodent cell lines which can account for theincreased cross-sensitivity to these agents.

The increased radiosensitivity of AT cells compared to normalcells has recently been associated with differences in cell cycleprogression for both cell types following X-irradiation (22). ATcells are reported to have less radiation- or bleomycin-induced

delay in the initiation of DNA synthesis compared to normal cells(22-24). Therefore, AT cells do not show the normal radiation-induced delay in progression from Gìto S or S to G2 (25-27).

FA fibroblasts, following treatment with HN2, showed no delayin S-phase progression (28), and HN2-treated V79/79 cellsshowed reduced S-phase delay (15). The above reports support

the idea of an association between increased cellular sensitivityand reduced G, and/or S-phase delay. However, results with the

human lymphoblastoma cell line TK6, which is sensitive to HN2,showed more S-phase delay than the less sensitive Raji cell line(29), and mouse lymphoma L5178Y cells showed an X-ray-

induced delay in DNA synthesis similar to that for resistant cells(20).

The present study was carried out to compare the sensitivitiesof a mouse myeloid leukemia cell line (ML-1) and normal mousebone marrow cells to X-ray-induced chromosome aberrationsand to examine the effects of X-rays on the cell cycle progression

of both cell types. Flow cytometric and Ml analyses were usedto study the effects of radiation on the cell cycle.

MATERIALS AND METHODS

Cell Culture. These studies used the ML-1 cell line derived from X-ray-induced RFM mouse myeloid leukemia (30) and normal mouse bone

marrow cells grown in vitro. As previously described by Au ef a/. (31,32), the ML-1 cell line, analyzed by the trypsin:Giemsa banding technique,

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was found to have a stable, diploid chromosome number of 40 and thefollowing consistent chromosomal alterations: deletion of chromosome2, Bands C-1 and F1-3; isochromosome 8; trisomy 19; and no Ychromosome. ML-1 cultures were maintained in RPMI 1640 (Gibco)containing 10% heat-inactivated(56°C,30 min)fetal bovine serum (FlowLaboratories)at 37°Cin an incubator humidified 95-100% in an atmosphere of 5% CO2 in air. Cultures were initiated at 4 x 10s cells/ml for

each experiment.Normal mouse bone marrow cell cultures were established prior to

each experiment using a modified version of the technique described byNakamura(33). Male RFM mice 9-11 wk old were sacrificed by cervicaldislocation, and the femurs were removed. Bone marrow cells wereflushed out of the femurs with Hanks' balanced salt solution (pH 7.0) at37°Ccontaining 5 units of heparin per ml using a 22-gauge needle.

Pooled bone marrow cells were centrifuged at about 350 x g for 4 minand resuspended in 37°CRPMI 1640 plus 25% heat-inactivated fetal

bovineserum. An aliquotof cellswas treated with ZapogloWnIIR(CoulterDiagnostics) to lyse the RBC, and WBC counts were determined usinga Coulter Counter (ModelZM; Coulter Electronics).The cell concentrationwas adjusted to approximately 3.5 x 106 nucleated cells per ml. Cellcultures were established with 7 x 106 nucleated cells per 2 ml ofmedium in 35-mm plastic culture dishes and incubated as described forthe ML-1 cells. It is appreciated that a small proportion of the cellsestablished in culture from the bone marrow will be represented by themetaphases analyzed; however, since the population analyzed is dividing, with a cycle time of about 20 h (Chart 1), it can be considered to besomewhat homogeneous.

Slide Preparation. For the cell cycle kinetics and chromosome aberration experiments, Colcemid(Sigma)at a final concentrationof 1 x 10"7

Mwas added 1 h prior to fixation. For the cell cycle kinetics, chromosomeaberration, and Ml experiments, cells were centrifuged, resuspended in10 ml of 0.075 Mpotassium chloride, and incubated at 37°Cfor 20 min.Cells were centrifuged, and approximately 1 ml of fixative (metha-nol:glacial acetic acid, 3:1) was slowly added to the pellet. Cells werewashed a total of 3 times with fixative, and singledrops of cell suspensionwere air dried onto wet microscope slides. Differential staining of thechromatids was performed using the method of Goto ef al. (34).

Determination of Cell Cycle Kinetics. An analysis of the cell cyclekinetics for each cell type was made to determine the time at which eachculture should be fixed in order to analyze metaphases that were in G,at the time of irradiation. To obtain the cell cycle kinetic data shown inChart 1, 10 MM BrdUrd was added at the time the cultures wereestablished, and the ML-1 cells were fixed 4,6, 8,10,12,18, 24, or 30h later. The normal bone marrow cells were fixed 6,12,18, 24, 30, 36,42, or 54 h later. The cell cycle kinetic data indicatethat, at the followingfixation times, 90% or more of the metaphases were in G, when theBrdUrd was added: 10-14 h after initiation for the ML-1 cells and 12-25

h after initiation for the bone marrow cells.To determine the time of irradiation which would allow both cell types

an equivalent length of time for repair of DNA damage before enteringthe S phase, the position of the G,-S border in the cell cycle wasdetermined as shown in Chart. 2. The ML-1 cells are a continuousculture, and the data from the experiment described above are replottedin Chart 2 to show the percentages of M1 and M1S cells. Because thenormal bone marrow cells are not a continuous culture, the followingprotocol was used to determine the time after irradiation that cells reachthe Gi-S border, for those G, cells which reach mitosis at 24 h. Normalbone marrow cells were fixed 24 h after being established in culture, andBrdUrd was added 4, 6, 8,10,12,18, or 24 h prior to fixation. That is,cells were in culture 20, 18, 16, 14, 12, 6, or 0 h before BrdUrd wasadded.

Slideswere preparedand differentiallystainedas previouslydescribed.At each time point, a minimum of 100 metaphases were analyzed todeterminetheir cell cycle stage at the time of BrdUrd addition.Cells wereclassified in a similar manner as described by Tice ef al. (35) into MO,M1S, M1, M2, M3, or greater, to indicate whether they were in G2,S, orGì,respectively,at the time BrdUrd was added.

X-lrradiation. For the chromosome aberration, Ml, and flow cytometrystudies, cell cultures were incubated 12 h before being irradiated with0.50, 1.0, 1.5, or 2.0 Gy of X-rays at 1 Gy/min. X-irradiations wereperformed with a 320-kVp Gemini industrial X-ray unit (TFI Corp., NewHaven, CT) operated at 250 kV and 12 mA with 2.3 mm of aluminumfiltration.

Chromosome Aberration Study. Cultures were fixed 12 and 14 hafter X-irradiation for the analysis of chromosome aberrations. Slideswere prepared and differentially stained as described previously. Onehundred metaphases with the M1 staining pattern were analyzed foreach X-ray dose, and all classes of aberrations were recorded. Theclasses of aberrations were reported as: chromosome-type aberrationswhich include dicentrics; rings; translocations; and deletions (both terminal and interstitial deletions). Chromatid-type aberrations include alltypes of deletions and exchanges. Acentric fragments associated withNUPD isochromatid deletions were included in the terminal deletioncategory.

Ml Determination. Giemsa-stainedslides were prepared for analysisof the Ml every 2 h from 0-14 h following irradiation with 0.5,1.0,1.5,or 2.0 Gy. For each sample, 2000 cells were counted, and the Ml wasexpressed as a percentageof the control.

Row Cytometry Study. A minimum of 1 x 108cells was fixed every2 h from 0-14 h postirradiation with 0.5,1.0, 1.5, or 2.0 Gy for the ML-1 cells and 2.0 Gy for the normal cells, for analysis by flow cytometry.The samples were prepared for analysis by the technique described byCrissmanef al. (36). Basically,each sample was resuspended in 2 ml of

10CH A

inUJVi5 60-a.

UJ

20-

36

(=••—j I—8:=(j_o-o—9*

52 6HOURS OF BrdU INCORR

100-1

18

Chart 1. Percentages of first (M1 ) (•)and second (M2) (O) division metaphasecells from in vitro normal mouse bone marrow cells (A) and mouse myeloid leukemia(ML-1) cells (8). TCis the average cell cycle time and ¡scalculated, as shown, fromthe point where 50% of the cells are in the ascending portion of the M1 curve tothe point where 50% of the cells are in the ascending portion of the M2 curve. 7efor the normal cells is 20 h, and Tc for the ML-1 cells is 15h. BrdU, BrdUrd.

HOURS OF BrdU INCORR

Chart 2. Percentage of metaphases from G, at the time BrdUrd was added (M1 )(•)and from S at the time BrdUrd was added (M1S) (O) for normal cells W andML-1 cells (B). The arrows indicate the X-ray treatment times at 12 and 14 h beforefixation. BrdU, BrdUrd.

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saline:EDTA at room temperature, and this solution was then added to5 ml of 95% ethyl alcohol (4°C)and refrigerated at 4°Cuntil analysis.

Cells were centrifugea for 10 min at 1500 rpm and then resuspended insaline:EDTA at 4°C. Pancreatic RNase (100 /¿g/ml)was added to each

cell sample, and solutions were left at room temperature for 30 min.Cells were stained with propidium iodide (50 /¿g/ml)for 10 min. Sampleswere passed through a 20-/im filter and then stored on ice until they

were analyzed. At least 50,000 cells per sample were analyzed for DNAcontent using an Ortho 50/H Cytofluorograph (Ortho Instruments, West-

wood, MA). For the normal bone marrow, cycling cells were selected foranalysis using a combined scatter versus fluorescence plot as describedby Mann ef al. (37). Cell cycle phase distributions were computed fromthe DNA histograms on a Digital Equipment POP 11/70 computer (DigitalEquipment Corp., Maynard, MA) using the method described by Fried(38).

RESULTS

Chromosome Aberrations. It is possible to obtain apparentdifferences in sensitivity to the induction of chromosome aberrations for different cell types, if the experimental protocol doesnot insure that for both cell types: (a) first division metaphasesare scored; (b) the time for repair of DNA damage betweentreatment and replication is equivalent (39). To this end, ananalysis of the cell cycle kinetics for both cell types was made.Chart 1 shows that the population of dividing normal bonemarrow cells analyzed in these experiments has a cell cycle timeof 20 h, and the ML-1 cells have a shorter cell cycle time of 15

h. In order to irradiate both cell types in Gìand allow each thesame length of time for repair of damage before entering the Sphase, a determination was made of the average time that ittook cells to pass from the G,-S border to metaphase as described in "Materials and Methods." Chart 2 shows that the

difference in the cell cycle times can be attributed primarily tothe longer Gìof the normal bone marrow cells. This finding is inagreement with other reports of cells with different cell cycletimes (40). From Chart 2 it can be seen that treatment of bothcell types 12 or 14 h before fixation would yield cells in mitosisthat were from the same region in Gì,approximately 6 or 8 h,respectively, before the midpoint of S.

The frequencies of the different types of aberrations are givenin Tables 1 and 2. The dose-response curves for combinedchromosome-type and chromatid-type aberrations are shown in

Table 1X-ray-induced chromosome aberrations in normal mouse bone marrow in vitro

Chromosome-type aberrations/cell

Table 2X-ray-induced chromosome aberrations in ML-1 cells

Chromosome-typeaberrations/cell

TreatmentControl12-h

harvest0.50 GyLOOGy1.50Gy2.00Gy14-h

harvest0.50 GyLOOGy1.50Gy2.00 GyNo.

of cellsanalyzed100100

100100100100

100100100Dicentrics0.00.04

±0.020.12 ±0.030.07 ±0.030.20 ±0.040.03

±0.020.06 ±0.020.13 ±0.040.23 ±0.05Terminal

deletions0.01±0.01s0.11

±0.030.19 ±0.040.33 ±0.060.29 ±0.050.06

±0.020.11 ±0.030.23 ±0.050.37 ±0.06Chromatid-type

aberrations/cell00.02

±0.010.00.01 ±0.010.02 ±0.010.01

±0.010.02 ±0.010.01 ±0.010.01 ±0.01a

Mean ±SE.

TreatmentControl12-h

harvest0.50Gy

1.00 Gy1.50Gy2.00Gy14-h

harvest0.50Gy

LOOGy1.50Gy2.00 GyNo.

of cellsanalyzed100100

100100

100100

100100100Dicentrics0.00.03

±0.020.06 ±0.020.10 ±0.030.19±0.040.03

±0.020.09 ±0.030.20 ±0.040.44 ±0.07Terminal

deletions0.01±0.01*0.26

±0.050.59 ±0.080.92 ±0.100.93±0.100.22

±0.050.62 ±0.080.64 ±0.080.80 ±0.09Chromatid-type

aberrations/cell0.00.11

±0.030.09 ±0.030.12 ±0.030.13±0.040.05

±0.020.12 ±0.080.11 ±0.030.09 ±0.03"

Mean ±SE.

Chart 3. Both cell types showed a dose-dependent increase inchromosome-type aberrations, with the ML-1 cells showing asignificantly greater sensitivity to the induction of chromosome-type aberrations at all X-ray doses and at both 12- and 14-htimes points (Chart 3). There was a 2-fold or greater increase interminal deletions in the ML-1 cells at all doses and time points

compared to the normal cells (Tables 1 and 2). Many of theseterminal deletions could be isochromatid NUPD deletions whichare indistinguishable from chromosome-type terminal deletions.

The terminal deletions contribute the most to the observedincrease in the chromosome-type aberrations for the ML-1 cells,

but there was a significant increase in the frequency of dicentricsat the 14-h fixation following 2.0 Gy of irradiation (0.44 for theML-1 cells versus 0.23 for the normal cells). The ML-1 cells, in

contrast to the normal cells, also had a significant number ofchromatid-type aberrations (deletions and exchanges). The truefrequency of chromatid-type aberrations is probably higher, be

cause isochromatid NUPD deletions were classified as chromosome-type terminal deletions as described in "Materials andMethods." The presence of many chromatid-type aberrations in

ML-1 cells irradiated in Gìindicates that the cells are progressing

into the S phase with unrepaired DNA damage that is then beingconverted into chromatid-type aberrations. Since both cell types

were treated at the same time prior to entering S, the presenceof only chromosome-type aberrations in the normal cells indicates: (a) that they are delayed in G, where misrepair of the X-ray-induced damage produces chromosome-type aberrations

and/or (b) that the normal cells have different DNA repair kineticscompared to the ML-1 cells. To investigate the first possibility,an analysis was made of the X-ray-induced perturbations of the

cell cycle.Mitotic Delay. Chart 4 shows the results of the Ml analysis.

Both cell types had a similar delay in the progression of cells intomitosis following irradiation, and this delay was more pronouncedwith increasing X-ray dose. For example, 2 h after irradiation ofthe ML-1 cells, the Ml decreased from 54% of the control at

0.50 Gy to 25% at 1.0 Gy, 15% at 1.5 Gy, and finally 0% at 2.0Gy. With time, the Ml of both cell types recovered to controllevels, and except for the normal cells at 1.50 Gy, the Mlexceeded the 100% level due to the temporarily delayed cellsprogressing through mitosis in a synchronous wave.

Flow Cytometry. Flow cytometry was used to further investi-

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Chromosome Type1.2-1 A. 12h

Chromolid TypeB. 12 h

I20-I

100-

-i D. 14h

20 1 2X-RAY DOSE (Gy)

Chan 3. Aberration frequencies per ceil following 0.5.1.0,1.5, or 2.0 Gy of X-ray. A and B present the results from the 12-h fixation time, and C and 0 presentthe results from the 14-h fixation lime. In/land C. the chromosome-type aberrationsare shown; in B and 0, the chromatid-type aberrations are shown for the normalcells (•)and the ML-1 cells (O). Points, mean; bars, SE.

gate the X-ray-induced perturbations of the cell cycle. Chart 5shows the DMA histograms for the ML-1 cells following X-

irradiation. The cell cycle phase distributions from these histograms were computer analyzed, and for comparative purposes,the control, 0.5-, and 2.0-Gy data are graphed in Chart 6. In

Chart 5, very little perturbation of the cell cycle is seen at thelowest dose of 0.5 Gy, but at increasing doses, there was a risein the G2-M peak which returned to normal at later times (data

not shown). The Ml at these time points was very low (Chart 4);therefore the increase in the G2-M peak is caused by G2 cells

which are being delayed in progressing into mitosis. Thus theflow cytometry data confirm the Ml data, showing a 62 delay inthe ML-1 cells following irradiation. The continued progression

of Gìcells into S, together with the inhibition of the progressionof G2-M cells through mitosis and back into Gì,resulted in adecrease in the proportion of cells in G,. For example, 6 h after

2 6 10 14 2 6 10 14 2 6 10 14 2 6 10 14HOURS AFTER IRRADIATION

Chart 4. Ml as percentage of control. Ml in A is after 0.5 Gy, 8 is after 1.0 Gy,C is after 1.5 Gy, and 0 is after 2.0 Gy of X-ray for the normal cells (•)and ML-1cells (O).

0.5 I

2469

HOURS AFTER IRRADIATION

Chart 5. RepresentativeONAhistograms showing cell cycledistributions of ML-1 cells 2, 4, 6,9, and 12 h after exposure to 0.5,1.0,1.5, or 2.0 Gy of X-ray. Theleft peak contains G, cells, the right peak contains G2-M cells, and the shadedregion contains the ¿-phasecells. For each individual histogram, the ordinate isthe frequency of cells, and the abscissa is the fluorescence.

irradiation with 2.0 Gy, the percentage of cells in G, was decreased to 28% of the total population (Chart 6). As cells continuethrough the cell cycle, there are fewer Gìcells moving into theS phase, resulting in a decrease in the proportion of S-phase

cells with time (Chart 6). Chart 5 shows that, as expected, thedecrease in the proportion of S-phase cells begins with the late-Gi-early S-phase cells. This is represented in the histogram bythe "dip" in the population of cells in the left-hand region of the

shaded S-phase area. As this population of cells passes through

the S phase, there is a subsequent decrease in the proportionof cells in late S phase of the cell cycle. These results along with

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80-,

6O-

40-

20-

•tì

/

G2/M

10 14 2 6 10 14 2HOURS AFTER IRRADIATION

10 14

Chart 6. The proportion of ML-1 cells in G,, S, GrM after 0 Gy (•),0.5 Gy (O),or 2.0 Gy (A) of X-ray. Points, average of three experiments; oars, SE.

the aberration data indicate that the ML-1 cells are progressing

without delay into the S phase with unrepaired DNA damagewhere misrepair of this damage can lead to chromatid-type

aberrations.Charts 7 and 8 show the DNA histograms and cell cycle phase

distributions, respectively, for the normal bone marrow cellsfollowing irradiation with 2.0 Gy. The X-ray-induced perturbation

of the cell cycle is not as dramatic in the normal bone marrowcells as it was in the ML-1 cells. The G2 delay indicated by the

Ml data is not evident in these DNA histograms (Chart 7). Withthe normal bone marrow cell cycle time being 20 h, only 2-3%of the cells occupy the Gz-M region, and the X-ray-induced Gi-and S-phase delays, discussed later, caused fewer cells toprogress into the G2-M phase. Therefore, any X-ray-induced G2delay would cause only a very small change in the G2-M peak,

and this will not be observed in the DNA histograms.In contrast to the ML-1 cells, which show a decrease in the

proportion of S-phase cells after irradiation, the proportion of

normal cells in the S phase increased with time after irradiation,indicating that the cells are delayed in their progression throughthis phase. From Chart 7, it appears that the accumulation oflate G,-early S cells is contributing the most to the overallincrease in the proportion of S-phase cells seen in Chart 8. These

results along with the aberration data indicate that the normalbone marrow cells are delayed in Gìand early S, where misrepairof the X-ray-induced damage can lead to chromosome-type

aberrations.

DISCUSSION

ML-1 cells, established from an X-ray-induced myeloid leukemia in an RFM mouse, showed an increased sensitivity to X-ray-

induced chromosome aberrations compared to in vitro normalmouse bone marrow cells. Specifically, G, irradiated ML-1 cellshad twice as many chromosome-type aberrations as did normalcells, many of which were in the terminal deletion category. ML-1 cells also had a significant number of chromatid-type aberrations, and the true frequency is probably higher since isochro-

matid NUPD deletions are included in the terminal deletion category. This is analogous to X-irradiated G, AT cells, which are

Chart?. Representative DNA histograms showing cell cycle distributions ofnormalcells 0,2,4,6,8,12, or 14 h after irradiation with 2.0 Gy of X-ray. For eachindividualhistogram, the ordinate is the frequency of cells, and the abscissa is thefluorescence. For definitions of peaks, see legend of Chart 5.

reported to have chromatid-type aberrations and a high fre

quency of fragments reported as terminal deletions (3,4,25,41,42).

Normal cells after treatment with a DNA-damaging agent havebeen shown to undergo an inhibition of DNA synthesis (22-24,43) and G,- and S-phase delays (25-27, 44, 45). RB cells, whichdo not have a greater sensitivity to X-ray-induced chromosomeaberrations and may or may not be sensitive to killing by X-rays(25, 46), also show a radiation-induced delay in the initiation of

DNA synthesis and a Gìblock (25). In contrast, AT cells do notshow the same radiation-induced inhibition of DNA synthesis orG,- and S-phase delays (22-27). Painter and Young (22) pro

posed that cell cycle delays may serve as a protective mechanism, which allows normal cells or cells with normal radiosensi-

tivity like RB cells additional time to repair DNA damage, andthat AT cells have increased radiosensitivity because they fail toundergo normal X-ray-induced cell cycle delays. However, this

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80

60

40o(Etua.

20

I I I j I I j10 »0 1414 2 6 10 14 2

HOURS AFTER IRRADIATIONChart 8. The proportion of normal cells in G,, S. and Gj-M after 0 Gy (•)and

2.00 Gy (A) of X-ray. Points, average of three experiments; bars, SE.

is not consistent with the increased S-phase delay in the HN2-sensitive TK6 cell line (29) or the similar X-ray-induced delay in

DMA synthesis in the mouse L5178Y cells (20), suggesting thatthere may be different mechanisms for the increased cellularsensitivity of these cells.

The results obtained by flow cytometry with the ML-1 and

normal bone marrow cells following irradiation indicate that thenormal cells are delayed in G, as well as S while the ML-1 cells

do not have the same G, and S delays. These results explainthe presence of chromatid-type aberrations in the ML-1 cells and

not the normal cells by showing that the normal cells undergo aGì-and S-phase delay following irradiation, which allows themextra time in Gn where misrepair of the X-ray-induced damageleads to chromosome-type aberrations. The ML-1 cells, like AT

cells, progress into the S phase with unrepaired DNA damagewhere misrepair of that DNA damage leads to chromatid-type

aberrations. There is the possibility that a repair defect exists inthe ML-1 cells, which would result in more DNA damage remain

ing when the cell enters the S phase, thus contributing to theformation of chromatid-type aberrations and the overall in

creased sensitivity observed for these cells. Additional experiments are in progress to analyze DNA repair kinetics for the ML-

1 and normal cells.There are conflicting reports concerning the relationship be

tween a G2 delay and cellular sensitivity. X-ray-sensitive L5178Ysublines have a greater X-ray-induced G2 delay than the parent

line (18,19). FA cells after mitomycin C treatment have a greaterG2 delay than normal cells (47), but HN2-treated FA cells have

less G2 delay (28). AT fibroblasts have been shown to have lessX-ray-induced mitotic delay (46, 48), while AT lymphoblastoid

cells do not show this decreased G? delay (27, 49). The Ml andflow cytometric analyses in this paper showed that the ML-1 andnormal cells have a similar dose-dependent delay in the progres

sion of cells from G2 into mitosis, and the Ml recovered in aboutthe same time after irradiation for both cell types.

We describe here a mouse myeloid leukemia cell line, ML-1,which has an increased sensitivity to X-ray-induced chromosome- and chromatid-type aberrations and decreased X-ray-

induced cell cycle delays compared to normal mouse bone

marrow cells. ML-1 cells, in response to radiation, have charac

teristics similar to irradiated AT cells, suggesting that these cellsmay have similar defects contributing to their increased radi-

osensitivities. In addition, it would be expected that these differences in aberration induction between the ML-1 and normal cells

would be paralleled by differences in cell killing, as for AT cellsversus normal human fibroblasts, since the majority of aberrations observed are cell lethal. It is also expected that there wouldbe similar differences in sensitivity between the two cell types tothe induction and transmission of non-cell-lethal aberrations. It is

proposed to determine from banded chromosome preparationsif there is a difference in the frequency of transmission of aberrations to subsequent cell generations in the ML-1 and normal

bone marrow cells. Experiments are also in progress to establishwhether other tumor cells have similar differences in X-ray sen

sitivity, cell cycle progression, and repair abilities.

ACKNOWLEDGMENTS

We would like to thank Dr. R. C. Mann for his helpful discussions and review ofthe manuscript. Dr. R. J. M. Fry for his critical review of the manuscript, and Dr. J.Fried for the use of the computer program for the analysisof DNA histogram cellcycle phase distributions.

REFERENCES

1. Higurashi, M., and Conen, P. E. In vitro chromosomal radtosensitivity in"chromosomalbreakage syndromes." Cancer (Phila.),32: 380-383,1973.

2. Taylor, A. M. R., Hamden, D. G., Artett, C. F., Harcourt, S. A., Lehmann, A.R., Stevens, S., and Bridges, B. A. Ataxia telangiectasia: a human mutationwith abnormal radiation sensitivity. Nature (Lond.),258: 427-429,1975.

3. Taylor, A. M. R., Metcalf, J. A., Oxford, J. M., and Harnden,D. G. Is chromatid-type damage in ataxia telangiectasiaafter irradiation at Goa consequence ofdefective repair? Nature (Lond.),260: 441-445,1976.

4. Taylor, A. M. R. UnrepairedDNA strand breaks in irradiated ataxia telangiectasia lymphocytes suggested from cytogenetic observations. Mutât.Res., 50:407-418,1978.

5. Taylor, A. M. R., Rosney, C. M., and Campbell, J. B. Unusual sensitivity ofataxia telangiectasiacells to bleomycin.Cancer Res., 39:1046-1050,1979.

6. Smith, P. J., and Paterson, M. C. Defective DNA repair and increased lethalityin ataxia telangiectasiacells exposed to 4-nitroquioline1-oxide. Nature(Lond.),287: 747-749, 1980.

7. Shiloh, Y., Tabor, E., and Becker, Y. Cellular hypersensitivity to neocarzino-statin in ataxia telangiectasia skin fibroblasts. Cancer Res., 42: 2247-2249,1982.

8. Sasaki, M. S., and Tonomura, A. A high susceptibility of Fanconi's anemia tochromosome breakage by DNA cross-linking agents. Cancer Res., 33:1829-1836,1973.

9. Cohen, M. M. The cytogenetic response of Fanconi's anemia lymphoblastoidcell lines to various clastogens. Cytogenet. Cell Genet., 34:230-240,1982.

10. Cleaver,J. E. Defective repair replication of DNA in xeroderma pigmentosum.Nature (Lond.),278: 652-656,1968.

11. Parrington, J. M., Delhanty, J. D., and Baden, H. P. Unscheduled DNAsynthesis, U.V. induced chromosome aberration, and SV40 transformation incultured cells from xeroderma pigmentosum.Ann. Hum. Genet., 35:149-160,1971.

12. Cleaver, J. E. Xeroderma pigmentosum: a human disease in which an initialstage of DNA repair is defective. Proc. Nati. Acad. Sci. USA, 63: 428-435,1969.

13. Sinclair, W. K. X-ray-induced heritable damage (small-colony formation) incultured mammaliancells. Radiât.Res., 21: 584-611,1964.

14. Fox, M., McMillan, S., Dun-ant,L. and Boyle, J. M. Relative sensitivity of V79and V79/79 cells to spontaneous and induced mutation to 6-thioguanine andouabain resistance. Mutât.Res., 95: 339-352,1982.

15. Graham, A., and Fox, M. The role of suppression of DNA synthesis andinhibition of cell cycle progression in cellular sensitivity to alkylation damage.Carcinogenesis(Lond.),4: 269-274,1983.

16. Scott, D., Fox, M., and Fox, B. W. The relationship between chromosomalaberrations, survival, and DNA repair in tumour cell lines of differential sensitivity to X-rays and sulphur mustard. Mutât.Res., 22: 207-221,1974.

17. Ehmann, U. K., Nagasawa, H., Petersen, D. F., and Lett, J. J. Symptoms ofX-ray damage to radiosensitive mouse teukemic cells: asynchronous populations. Radiât.Res., 60:453-472,1974.

18. Rosenberg,H. M., Robinson,J. K., Horng, M. F. L., Gregg, E. C., and Nygaard,

CANCER RESEARCH VOL. 45 NOVEMBER 1985

5326

on June 29, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

X-RAY SENSITIVITIES OF LEUKEMIC AND NORMAL CELLS

0. f. Sensitivity to X-rays in terms of mitotic delay (Sd) and killing (Sk):correlation between Sd and Sk for subîmesof murine leukaemic cells. Int. J.Radiât.Brol.,29: 197-200,1976.

19. Scott, D., and Zampetti-Bossesler, F. The relationship between cell killing,chromosome aberrations, spindle defects, and mitotic delay in mouse lym-phoma cells of differential sensitivity to X-rays. Int. J. Radiât.Bioi., 37:33-47,1980.

20. Ockey, C. H. Differences in replicónbehaviour between X-irradiationsensitiveL5178Y mouse lymphoma cells and ataxia telangiectasia fibroblasts usingDNA fiber autoradiography. Radiât.Res., 94:427-438,1983.

21. Fox, M., and Fox, B. W. The establishment of cloned cell lines from Yoshidasarcomas having differential sensitivities to méthylènedimethane sulphonatein vivo and their cross-sensitivity to X-ray, UV, and other alkylating agents.Chem.-Biol. Interact., 4: 363-375,1971/1972.

22. Painter, R. B., and Young, B. R. Radiosensitivity in ataxia telangiectasia: anew explanation. Proc. Nati. Acad. Sci. USA, 77; 7315-7317,1980.

23. Cramer, P., and Painter, R. B. Bleornycin-resistant DNA synthesis in ataxiatelangiectasiacells. Nature (Lond.), 297:671 -672,1981.

24. Jaspers, N. G. J., de Wit, J., Regulski, M. R., and Bootsma, D. Abnormalregulation of DNA replication and increased lethality in ataxia telangiectasiacells exposed to carcinogens. Cancer Res., 42: 335-341,1982.

25. Nagasawa, H., and Little, J. B. Comparison of kinetics of X-ray-induced cellkilling in normal, ataxia telangiectasia, and hereditary retinoblastoma fibroblasts. Mutât.Res., 109: 297-308,1983.

26. Nagasawa, H.. Latt, S. A., Lalanae, M. E., and Little. J. B. Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations,and cell killing in ataxia telangiectasia (AT) fibroblasts. Mutât.Res., 748: 71-82, 1985.

27. Imray, F. P., and Kidson, C. Perturbations of cell-cycle progression in X-irradiated ataxia telangiectasia and Huntington's disease cells detected by

DNA flow cytorneine analysis. Mutât.Res., 112: 369-382,1983.28. Dean, S. W., and Fox, M. Investigation of the cell cycle response of normal

and Fanconi's anaemia fibroblasts to nitrogen mustard using flow cytometry.J. Cell Sci., 64: 265-279,1983.

29. Dean, S. W., and Fox, M. DNA repair, DNA synthesis, and cell cycle delay inhuman lymphoblastoid cells differentially sensitive to the cytotoxic effects ofnitrogen mustard. Mutât.Res., 732:63-72,1984.

30. Upton, A. C., Jenkins, V. K., and Conklin,J. W. Myeloid leukamiain the mouse.Ann. NY Acad. Sci., 714: 189-202,1964.

31. Au, W. W., Luippold, H. E., and Often, J. A. Developmentof a transplantablemouse myeloid leukemia model system: a preliminary report. Prog. NucleicAckJRes. Mol. Bid., 29: 47-50,1983.

32. Au, W. W., andGoldenthal,K. L. Normalsister chromatidexchangefrequenciesduring growth of a transplantable murine myekjid leukemia. Cancer Genet.Cytogenet., 14:125-130,1985.

33. Nakamura, K. The proliferation of plasma cells from mouse bone marrow in

vitro. J. Exp. Med., 735:476-479,1972.34. Goto, K., Maeda, S., and Sugiyama,Y. Factors involved in differential Giemsa-

staining of sister chromatids. Chromosoma (Beri.),66: 351-359,1978.35. Tice, R., Schneider,E. L, and Rary,J. M. The utilization of bromodeoxyuridine

incorporation into DNAfor the analysisof cellular kinetics. Exp. Cell Res., 702;232-236,1976.

36. Crissman, H. A., and Steinkamp,J. A. Rapid,one-step staining procedures foranalysis of cellular DNA and protein by single and dual laser flow cytometry.Cytometry, 3: 84-90,1982.

37. Mann, R. C., Popp, D. M., and Hand, R. E., Jr. The use of projections fordimensionalityreductionof flow cytometric data. Cytometry, 5:304-307,1984.

38. Fried, J. Method for the quantitative evaluation of data from flow microfluo-rometry. Comput. Biomed. Res., 9: 263-276,1976.

39. Preston, R. J. The induction of chromosome aberrations in germ cells: adiscussion of factors that can influence sensitivity to chemical mutagens. In:T. Sugimura, S. Kondo, and H. Takebe (eds.), Environmental Mutagens andCarcinogens,pp. 463-474. Tokyo: Universityof Tokyo Press, 1982.

40. Prescott, D. M. Reproductionof Eukaryotic Cells, New York: Academic Press,1976.

41. Natarajan, A. T., and Meyers, M. Chromosomal radiosensitivity of ataxiatelangiectasiacells at different cell cycle stages. Hum. Genet., 52:127-132,1979.

42. Bender, M. A. Relationshipof DNA lesions and their repair to chromosomalaberration production. In: W. M. Generoso, M. D. Shelby, and F. J. de Serres(eds.), DNA Repair and Mutagenesis in Eukaryotes, pp. 245-265. New York:Plenum Press, 1980.

43. Watanabe, M., Takabe, Y., Katsumata, T., Terasima, T., and Umezawa, H.Response in macromotecular synthesis of mouse L-cells to bleomycin, withspecial reference to cell-antibiotic interaction. J. Antibiot. (Tokyo), 26: 417-423,1973.

44. Leeper, D. B., Schneiderman,M., and Dewey,W. C. Radiation-induceddivisiondelay in synchronized Chinese hamster ovary cells in monolayer culture.Radiât.Res., 50: 401-417,1972.

45. Gemer, E. W. DNA replication in X-irradiated Chinese hamster ovary cells.Radiât.Res., 77: 387-397,1977.

46. Zampetti-Bosseler, F., and Scott, D. Cell death, chromosome damage, and-mitotic delay in normal, ataxia telangiectasia, and retinoblastoma fibroblastsafter X-irradiation. Int. J. Radiât.Bid., 39: 547-558,1981.

47. Kaiser, T. N., Lojewski, A., Dougherty, C., Juergens, L., Sahar, E., and Latt,S. E. Flow cytometric characterization of the response of Fanconi's anemiacells to mitomycin C treatment. Cytometry, 2: 291-297,1982.

48. Scott, D., and Zampetti-Bosseler,F. Cell cycle dependenceof mitotic delay inX-irradiated normal and ataxla-telangiectasiafibroblasts. Int. J. Radiât.Bid.,42: 679-683,1982.

49. Ford, M. D., Martin, L., and Lavin, M. F. The effects of ionizing radiation oncell cycle progression in ataxia telangiectasia. Mutât.Res., 725: 115-122,1984.

CANCER RESEARCH VOL. 45 NOVEMBER 1985

5327

on June 29, 2018. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1985;45:5321-5327. Cancer Res   Marilyn J. Aardema, William W. Au, Russell E. Hand, Jr., et al.   Chromosome Aberrationsand Normal Mouse Bone Marrow Cells to X-Ray-induced Differential Sensitivity of a Mouse Myeloid Leukemia Cell Line

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