characterization of late-arising chromosome aberrations in human b-cell lines established from...

Cancer Genetics and Cytogenetics 187 (2008) 112e124

Characterization of late-arising chromosome aberrations in humanB-cell lines established from a-raye or g-rayeirradiated lymphocytes

Kimio Tanakaa,*, Tirukalikundram S. Kumaravelb, Shozo Ihdab, Nanao Kamadac

aDepartment of Radiobiology, Institute for Environmental Sciences, Rokkasho, Aomori 039-3213, JapanbResearch Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi, Minamu-ku, Hiroshima 734-8552, Japan

cHiroshima Atomic Bomb Relief Foundation, Kurakake, Asakita-ku, Hiroshima 739-1743, Japan

Received 13 June 2008; received in revised form 29 July 2008; accepted 7 August 2008

Abstract To clarify the characteristics of late-arising (de

* Corresponding

1982.

E-mail address: k

0165-4608/08/$ e see

doi:10.1016/j.cancerg

layed) chromosome aberrations after irradiation inhuman lymphocytes, 30 B-cell lines were established from the peripheral blood from a healthy adultdonor, the lymphocytes of which were exposed to a-rays or g-rays and then used for experiments.Chromosome aberrations were serially observed at several passages by both conventional cytogeneticsand fluorescence in situ hybridization analysis using subtelomere probes. These B-cell lines derivedfrom lymphocytes with a history of radiation exposure had higher percentages of delayed chromosomeaberrations, such as dicentrics, rings, endoreduplication, hyperdiploid, hyperploidy, and telomere as-sociation. Furthermore, a-ray exposure induced higher chromosome instability than g-ray exposure,indicating that delayed chromosome aberrations were related with radiation quality. Chromosomeinstabilities were also observed at the subtelomere. Cell lines showing high chromosome instabilityhad high DNA-PK activity, low expressions of Ku70, p53, and TRF1 proteins after stimulation withradiation. These results indicate that mechanisms underlying delayed chromosome aberrations mightbe epigenetic, and multiple factors such as defects of DNA-PK, subtelomere, and telomere might beassociated. � 2008 Elsevier Inc. All rights reserved.

1. Introduction

The ‘‘mutation theory’’ of carcinogenesis, in whichunrepaired, radiation-induced DNA damage occurring inoncogenes or any other genes is fixed as a mutation, andmutated genes accumulate for a long time, might be associ-ated with the development of cancer. Several studies,however, do not seem to support this concept [1e7]. Radia-tion-induced genomic or chromosome instability is used asa term to describe the increase of new aberrations, long afterirradiation. These late-arising (delayed) aberrations after ir-radiation have been found in various types of endpoints, in-cluding delayed cell death [8], delayed mutation [9],delayed chromosome aberrations [10e12], and delayed celldifferentiation [13,14]. Radiation-induced genomic instabil-ity with respect to the chromosome aberration endpoint wasfirst demonstrated in one-cell mouse embryos with x-ray irra-diation and in mouse fetal cells [15], as well as in mousehematopoietic stem cells after high linear electron transfer

author. Tel.: þ81-175-71-1754; fax: þ81-175-72-

[email protected] (K. Tanaka).

front matter � 2008 Elsevier Inc. All rights reserved.

encyto.2008.08.009

(LET) radiation [6]. These studies were later confirmed byseveral reports using other rodent-cultured cells and severalhuman cultured cells after low and high LET irradiation[11,12,16e22]. Genomic instability was also detected ingerm cells of irradiated parents, especially males, and inthe offspring born to them [23].

Studies on chromosome instability were performedmostly in rodent cells. At this point, however, the questionis raised as to whether chromosome instability occurs inhuman tissues after irradiation, because rodent culturedcells originally have a feature of much higher chromosomeinstability than human cells. Our previous study alsorevealed that human bone marrow cells and lymphocytesfrom a normal adult had 0.5e2.3 and 0.7e1.3% aberrationsafter 241Am a-ray exposure, respectively. On the contrary,mouse bone marrow cells had 15e37% chromosome aber-rations in CFU-A bone marrow colonies [6].

Furthermore, so far we have not had enough evidence toconfirm the relationship between chromosomal instability in-duced in the late stage after radiation exposure and cancer de-velopment in human tissues. To elucidate the characteristicsof radiation-induced chromosome instability, we observedthe chromosomal instability in human B-lymphocyte clones

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113K. Tanaka et al. / Cancer Genetics and Cytogenetics 187 (2008) 112e124

established from irradiated lymphocytes at several tens ofdays to a longer time period using several chromosome tech-niques. These results suggested that delayed chromosomeaberrations were not directly induced by radiation. The cyto-genetic and molecular characteristics of the B-cell lines thatcould survive were totally different from those soon afterexposure. This information will be helpful to confirm thisnew hypothesis in human tissues, that cancer does not de-velop directly from radiation-induced mutation, but rather in-directly, through genomic or chromosome instability thatoccurs after radiation exposure.

2. Materials and Methods

2.1. Radiation sources

A 241Am a-ray source, installed at the Radiation BiologyCenter, Kyoto University, was used for the present study. Theoriginal dose was 36 MBq, the mean energy was E 5

2.47MeV, and the LET was 169.1 keV/mm. Radiation dosesof 0.125 and 0.25 Gy were used for the present study. Assum-ing that the diameter of the lymphocyte nucleus is 6 mm, themean radiation dose rate was 180 mGy/sec. Approximately30 mL of lymphocytes suspended in RPMI medium waspoured on 2.0 � 2.0-mm dishes and a Mylar membrane(Millipore, Bedford, MA) dabbed on the cell suspension tomake a single-cell layer. The irradiated cell suspension wasrecovered by washing out the RPMI medium and then col-lected into a 15 mL tube. Two or three plates were used forone radiation dose point. After the exposed cell suspensionshad been washed four times with phosphate-buffered saline(PBS), short- and long-term cultures were set up. The relativebiologic effectiveness of 241Am a-rays was calculated as 3.9relative to 60Co g-rays on the basis of the lymphocyte chro-mosomes observed (data not shown).

A 60Co g-ray source at the Research Institute for Radi-ation Biology & Medicine, Hiroshima University, was used.60Co g-ray doses measured with an HU-5 chamber, whichis the tertiary standard dosimeter (Japanese Association ofRadiological Physicists dosimeters). The g-irradiation ofisolated lymphocytes was carried out using 60Co (111TBq) at a rate of 200 mGy/min at the g-irradiation facilityof Hiroshima University (Isotron RTGS-21; ShimadzuCorp., Kyoto, Japan). The irradiation time was about 100minutes for the 2-Gy group and 200 minutes for the 4-Gygroup. All irradiations to human lymphocytes were per-formed at 37�C.

2.2. Establishment of B-cell lines

About 2 � 107 G0 lymphocytes were separated by Ficollsedimentation from 10 mL of blood drawn from a healthyadult. After irradiation, the cells were washed two timeswith PBS solution, infected with Epstein-Barr virus(EBV) for 12 hours, and cultured for 1 week in a 50-mLtube. The cell solution (5 mL) from the growing cells

was again cultured in a 76-well microplate for severalmonths. The growing colonies were isolated and transferredto 4 wells of a microplate for 1e2 months for furtherexpansion. The growing colonies were separated from asa cell line and cultured in 50-mL culture flasks to obtain2 � 107 cells by a further expanded culture. Half of themedium was changed every cell passage. Finally, 21 B-celllines were established from G0 lymphocytes irradiated with0.5 or 1.0 Gy of 60Co g-rays and one cell line from 0.125Gy of 241Am a-rays. Eight B-lymphocyte cell lines werealso established from non-irradiated lymphocytes fromthe same person, for a control study. Established B-celllines were irradiated with 0.5, 1.0, 2.0, 3.0 and 4.0 Gy of60Co g-rays (200 mGy/min), and the radiation sensitivitywas evaluated by scoring the rates of unstable-type chromo-some aberrations in 200 metaphases. The cell lines werealso irradiated with 2 Gy of 60Co g-rays at the dose ratesof 300 or 900 mGy/min, and protein expressions were mea-sured to determine the alteration of cellular response andsister chromatid exchange (SCE) in their cellular response.

2.3. Chromosome and fluorescence in situ hybridization(FISH) analyses

Chromosomes were analyzed by conventional Giemsastaining and G-banding with trypsin. The karyotype was deter-mined according to ISCN 1995 [24]. The chromosome aberra-tions of these cell lines were analyzed serially at about every10 passages. Chromosome or interphase/metaphase FISHanalyses using centromere probe of chromosome 12 (Oncol,Gaithersburg, MD), three subtelomere probes (Tel 1q, Tel 3p,and Tel 7q DNA probes; Oncol), telomere probes (Cambio,Cambridge, UK), and whole painting Y chromosomeprobes (Abbott Molecular/Vysis, Dawners Grove, IL) wereperformed at about every 20 passages. These probes wereapplied according to the manufacturer’s recommendations.

2.4. SCE analysis

Three B-cell lines (T13-1, T8-1, and T14-1) derivedfrom 241Am a-, 60Co g-, or non-irradiated lymphocytes,respectively, were reirradiated with 60Co g-rays at 200mGy/min. We analyzed the cell cycle in the three cell lineswith the fluorescent plus Giemsa (FPG) method [25]. Aftersetting up the culture, the cells were cultured for 72 hours.BrdU (5 mg /mL) was added and cultured for 24 hours andharvested at 50 hours. Chromosome slides were stainedwith 33258 Hoechst, exposed to ultraviolet light, andstained with Giemsa. Fifty metaphases were scored underthe microscope. The populations of first and second mitotic(M1 and M2) cell were evaluated.

2.5. Western blot analysis

Protein expressions of Ku70, p53, p21, and TRF1 wereanalyzed by Western blotting and immunostaining.

114 K. Tanaka et al. / Cancer Genetics and Cytogenetics 187 (2008) 112e124

The protein amount was measured by soft program in a con-forcal laser scanning microscope (Olympus, Tokyo, Japan).B-lymphocyte cells derived from five cell lines with orwithout chromosome instability were washed twice withPBS and lysed in 0.5 mL RIPA buffer (1% Nonidet-P40),0.5% sodium deoxycholate, 0.1% SDS, 50 mg PMSF in iso-propyl alcohol, 50 mg of 100 mmol/L sodium orthovana-date, and 15 mL aprotonin in PBS (pH 7.4). The lysatewere cleared by centrifugation at 15,000 rpm at 4�C for20 minutes. The protein contents of the cell lysate weremeasured using the DC protein estimation kit from BioRad (Hercules, CA). A volume corresponding to 20 mg ofcell lysate was mixed with an equal volume of samplebuffer [2% SDS, 10% glycerol, 60 mmol/L Tris-HCl (pH6.7), 5% mercapto-ethanol, and 0.01% bromophenol blue]and denatured at 100�C for 90 seconds. The denatured sam-ples were resolved on a 10% SDS polyacrylamide gel, andthe proteins were transferred to a nitrocellulose membrane(Hybond ECL; Amersham, Arlington Heights, IL). Theblots were incubated with blocking buffer [5% bovineserum albumin (BSA) in PBS-T; Santa Cruz BiotechnologyInc., California, USA] for 1 hour at room temperature. Theblots were rinsed in PBS-T and incubated with anti-mouseantibody conjugated with horseradish peroxidase (1:3,000dilution in PBS-T; Santa Cruz Biotechnology Inc., SantaCruz, CA). The blots were then rinsed in PBS-T and theantigeneantibody conjugates were visualized usingenhanced chemiluminescence (ECL; Amersham) accordingto the manufacturer’s recommendation.

2.6. Immunofluorescent staining

B-lymphocytes cells from eight B-cell lines with andwithout chromosome instability were analyzed by immuno-fluorescnece staining for the expression and localization ofKu70, p53, and TRF1 proteins. All antibodies wereobtained from Santa Cruz Biotechnology and were usedwithin 3 months of the date of purchase. The fixed slideswere washed with PBS, treated with acetone, blocked with10% normal serum, and incubated with Ku70, p53, andTRF1 antibodies (2 mg/mL) at 37�C in a humidified incu-bated for 60 minutes. The slides were washed twice withPBS at room temperature and incubated with FITC-labeledanti-goat IgG (10 mg/mL; Santa Cruz Biotechnology) for 30minutes at 37�C. Following three washes with PBS, thepreparations were mounted in antifade (Oncor, Gaithers-burg, MD). Appropriate negative controls were performedsimultaneously.

Cytospin slides were prepared at various time points(500 rpm for 3 minutes; Shandon Inc., Pittsburgh, PA)and air-dried. The slides were washed briefly in PBS, fixedin methanol (30�C for 30 minutes), and finally immersed inice-cold acetone for 5 seconds for proper penetration ofantibodies. The fixed slides were washed with PBS, treatedwith RNase (0.1 mg/mL) for 20 minutes, blocked with 10%BSA, and incubated with Ku 70, p53, and TRF 1 antibodies

(2 mg/mL) at 37�C in a humidified incubator for 60minutes. The slides were washed three times with PBS atroom temperature and incubated with FITC-labeled anti-goat IgG (10 mg/mL; Santa Cruz Biotechnology) for 30minutes at 37�C. After three washes with PBS, the prepara-tions were mounted in antifade (Oncor) containing 0.001mol/L propidium iodide (PI; Sigma, St Lois, MO).

2.7. Immunofluorescence microscopy

The slides were scanned under a laser-scanning micro-scope LSM-GB200 (Olympus Optical Inc., Tokyo, Japan),and the immunofluorescence images were taken and storedon an IBM computer. Analysis and mapping of signal inten-sity were performed using the application soft program ofLSM-GB200. The Ku70, p53, and TRF1 antibodiesproduced a green fluorescence, and the nucleus stainedred due to the PI.

2.8. Measurement of DNA-PK activity

Activity was evaluated by a filter-binding assay. TheDNA-dependent protein kinase (DNA-PK) peptidesubstrate is a highly specific substrate for DNA-dependentprotein kinase having the sequence Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys–Lys. DNA-PK isa nuclear serine/threonine protein kinase that, when acti-vated by DNA, phosphorylates several DNA-binding sub-strates, including the tumor suppressor protein p53, thesimian virus 40 (SV-40) large T antigen, and several tran-scription factors (e.g., SP1, OCT-1, c-Fos, and serum re-sponse factor). B-cell nuclear extract was prepared for theDNA-PK assay. To use the B-cell nuclear extract as a sourceof DNA-PK, it is necessary to first remove the endogenousDNA from the extract. To remove endogenous DNA, weapplied an aliquot of the B-cell line nuclear extract to 2mL of DEAE Sepharose, Fast Flow, pre-equilibrated inbuffer A (100 mmol/L KCl, 50 mmol/L Hepes, 0.2mmol/L EGTA, 0.1 mmol/L EDTA, and 1.0 mmol/LDTT), washed with 10 mL buffer A, and then eluted the en-zyme with 4 mL buffer B (400 mmol/L KCl, 50 mmol/LHepes, 0.2 mmol/L EGTA, 0.1 mmol/L EDTA, and 1.0mmol/L DTT). The reaction contained DNA-PK activationbuffer, DNA-PK 5� reaction buffer, DNA-PK biotinylatedpeptide substrate, BSA, and [g-32P]ATP mix, which wasprepared in 0.5e1.0 mL microcentrifuge tubes. For thecontrol reaction, DNA-PK control buffer, a mix of DNA-PK 5� reaction buffer, DNA-PK biotinylated peptide sub-strate, BSA, and [g-32P]ATP was used. They were mixedwell at 30�C for 1e5 minutes. Enzyme dilution bufferwas prepared by distilled water with added BSA to a finalconcentration of 0.1 mg/mL. Enzyme samples were dilutedtwofold to 1:16.

We initiated the reaction by adding the appropriateamount of enzyme sample (0e9.95 mL) and then adjustedthe reaction to a final volume of 25 mL using deionized


water. It was incubated at 30�C for 5 minutes. The reactionwas terminated by adding 12.5 mL of termination buffer toeach reaction and mixed well. The terminated reactions canbe kept at room temperature during processing and arestable at 4�C for at least 24 hours. Each terminated reactionwas spotted onto a preincubated square of the SAM2TM

membrane. After all samples had been spotted, we followedthe wash procedure and rinse steps. The reaction tube wassaved for the next step. The membrane squares containingsamples from the previous step were put into a washingcontainer. Washes were done with an orbital platformshaker set on low as follows: wash 1� for 30 seconds with200 mL of 2 mol/L NaCl, 3� for 2 minutes each with 200mmol/L of 2 mol/L NaCl, and wash 4� for 2 minutes eachwith 200 mL of 2 mol/L NaCl in 1% H3PO4 , and wash2� for 30 seconds, each with 100 mL of deionized water.

The membrane was dried on a piece of aluminum foilunder a heat lamp for 5e10 minutes or air-dried at roomtemperature for 30e60 minutes. Total counts for the spe-cific activity of [g-32P]ATP were made. The SAM2TM

membrane was separated by square using forceps, placedin the scintillation vials, and counted. The specific activityof [g-32P]ATP in cpm/pmol was calculated as [37.5/5 �(average counts/minute) divided by 2,500. The enzymaticactivity of DNA-PK can be determined by subtracting theactivity of the enzyme in the absence (control buffer) fromthat of the enzyme in the presence of an activator (activa-tion buffer). Enzyme-specific activity in pmol ATP/min-ute/mg of protein was calculated.

2.9. Polymerase chain reactionesingle-strandconformation polymorphism (PCR-SSCP) analysis of theTrp53 gene mutation

DNA was isolated according to standard procedure.Oligonucleotide primers for exons 5e8 of the Trp53 tumorsuppressor gene were synthesized. All primers includeda portion of intron to avoid amplification of the Trp53pseudogene. PCR-SSCP analysis was done according tostandard procedure. Briefly, primers were end-labeled with[g-32P]ATP using the T4 polynucleotide kinase. GenomicDNA was amplified for 30 cycles in 10 mL of a reactionmixture containing 100 ng of template DNA, 4 mM end-la-beled primers, 200 mM each of dNTP (dATP, dCTP, dGTP,and dTTP), and 0.05 units of Taq DNA polymerase. Eachcycle consisted of 94�C for 1 minute, 52�C for 1 minute,and 72�C for 30 seconds. Reaction mixtures were treatedwith 10 mL of 95% formamide, 20 mmol/L EDTA, 0.05%bromophenol, and 0.05% xylene cyanol, and then dena-tured at 90�C for 2 minutes. An aliquot (1 mL/lane) wasapplied to a 6% nondenaturing polyacrylamide gel contain-ing 5% glycerol, and then electrophoresed at 30e40 wattsfor 2e3 hours at room temperature with fan cooling. Thegel was dried and x-ray film was exposed. Putative mutantbands of the Trp53 gene were detected by PCR-SSCPanalysis.

2.10. In situ telomeric repeat amplification protocol(TRAP) assay

The amount of telomerase in each cell was measured byin situ TRAP assay. Cell preparations of 100 mL fromB-cell lines were made with Cytospin (Shandon Inc.).The fresh slides were immediately used for in situ TRAPassay. Slides were washed by 1� PBS for 5 minutes, eachtwo times, and 1� PBS þ 50 mmol/L MgCl2 for 5 minutesone time at room temperature. The slide was incubated30�C for 30 minutes with 2.0 mL of FITC-labeled TSprimer and TRAP assay mixture solution (5.0 mL of 10�TRAP buffer, 1.0 mL of 50� dNTP mix, and 39.6 mL of dis-tilled water) in an 18�18-mm area. The area was shieldedby manicure oil and covered with paraffin. The 2 mL ofFITC-labeled CX primer and 0.4 mL of Taq polymerase(5 U/mL) were then added on the area. The total volumeof the solution in the 18 � 18 mm area was 50 mL. The18 � 18-mm coverslip was added and shielded by manicureoil. The slide was put on a PCR machine (TAITEC, Fukuo-ka, Japan) and applied to PCR reaction. The reaction was1.5 minutes at 90�C (one time), and repeated for 30 cyclesof 94�C for 30 second, and 60�C for 30 seconds by the ther-mocycler. After the reaction, the shielded coverslip wasremoved and washed with distilled water one time in thecaplin jar. The slide was then stained with PI and observedwith a fluorescent microscope using appropriate elimina-tion and excitation filters. The amount of FITC-labeledtelomerase was measured with a laser-scanning microscope(Olympus Optical Inc., Tokyo, Japan).

3. Results

3.1. Cytologic characterizations of established B-celllines

We were able to establish 30 B-cell lines (1, 21, and 8B-cell lines from 241Am a-rayeexposed, 60Co g-rayeexposed, and nonexposed lymphocytes, respectively). Eigh-teen sublines were also established. Mean establishmenttimes for the B-cell lines from starting the culture infour-well microplates ocurred 40 and 45 days after 241Ama-ray and 60Co g-ray exposure, in contrast to 23 days innonexposed B-cell lines, which is known as cell divisiondelay after irradiation (Table 1). The successful percentagefor establishment and the establishment time of EBV-stim-ulated B-cell lines were clearly influenced by radiationquality. Most EBV-stimulated B-lymphocytes establishedfrom 241Am a-rayeirradiated lymphocytes did not showrapid growth and died within 1 month, except for one cellline (T13-1). Slow cell growth of the T13-1 cell line from241Am a-rayeirradiated lymphocytes was confirmed bythe FPG method. The percentages of first mitosis (M1), sec-ond mitosis (M2), and third mitosis (M3) of T14-1 and T8-1, which were derived from nonirradiated and g-rayeirradiated lymphocytes, were 18, 64, and 8%, and 21, 65,

Table 1

Characteristics in 30 B-cell lines and 18 sublines established from 60Co g-raye and 241Am a-rayeirradiated lymphocytes and nonexposed lymphocytes

Radiation

source

Establishment

time mean days

No. of established

cell lines (No. of

sublines)

No. of cell

line with

chromosome

instability (No.

of sublines)

No. of cell

line with high

spontaneous

chromosome

aberrations

No. of cell

lines with growth

retardation

No. of cell

lines with telomere

association

No. cell

lines with

subtelomere

instability

Not exposed 23 8 (6) 0 (0) 0 (0) 0 0 060Co g-ray 45 21 (6) 5 (1) 5 (0) aee 1 b 1 b 2 b,e

241Am a-ray 40 1 (6) 1 (6) 1 f(6) 1 f(6) 1 f(6) 1f(2)

aef Each cell line named as T8-1,T8-8, T8-2,T8-4,T7-3, and T13-1.


and 14%, respectively, which were almost same, but inthe T13-1 cell line derived from a-rayeirradiatedlymphocytes, the percentages were 76, 48, and 0%. Nocells from the T13-1 cell line entered into M3 phase. Thismeant that growth of the T13-1 cell line was slow. Allestablished B-cell lines were maintained for up to at least10 passages to analyze chromosome instability and cellviability.

In the present analysis, cell lines with high chromosomeinstability were defined as having higher rates of spontane-ous chromosome aberration (more than 10%), and havingeither one of spontaneous cell growth, cell death, telomerefusion, or subtelomere instability. Spontaneous chromo-some aberrations were classified as unstable-type aberra-tions (e.g., dicentrics, rings, fragments, hyperdiploidy, andhyperploidy) and chromatid-type aberrations (e.g., chroma-tid breaks, gaps, and exchanges, and endoreduplication),most of which are not observed soon after irradiation inG0 fresh lymphocytes. On the other hand, dicentrics, rings,and fragments were induced by radiation, but they wereeliminated at each cell division after irradiation. Six ofthe 30 B-cell lines were classified as having high chromo-some instability (Table 1).

3.2. Delayed chromosome aberrations in B-cell lines

Fig. 1 shows the yields of chromosome aberrations ina cell line (T13-1) established by 241Am a-ray exposureand a nonexposed cell line (T14-1) at passages 1e5. TheT13-1 cell line had a higher percent of spontaneous aberra-tions (9.6e18.6% in passages 1e5) than the T14-1 controlcell line (0.8e6.4% in passages 1e5). Different types of chro-mosome aberrations appeared continuously at each passage.Among the three cell lines, the T8-8 B-cell line, developed us-ing 0.5 Gy of 60Co g-rays, had the highest chromosomal in-stability at passages 1e9 (5.2e36.8%), with dicentrics andrings, chromatid-type aberrations, hyperdiploidy, endoredu-plication, and telomere fusion (Fig.2).

Three cell lines (T13-1, T8-1, and T14-1) were kept in theculture up to passage 117, to sequentially observe chromo-some aberration frequencies. Half of the media were changedevery cell passage (once a week). In the T13-1 and T8-1 celllines established from 241Am a-raye and 60Cog-rayeirradiated lymphocytes, respectively, 9.6e58.9%

and 12.8e81.0% of cells had spontaneous chromosome aber-rations at passages 1e117, respectively (Table 2). These twocell lines had much higher (spontaneous chromosome aberra-tions) chromosome instability than the T14-1 cell linederived from nonexposed lymphocytes (0.8e8.0% inpassages 1e117). Frequencies of chromosome 12 trisomywere higher in T13-1 and T8-1 cell lines than in the T14-1 cellline. All the cell lines developed clonal aberrations in thepassages.

3.3. Delayed chromosome aberrations at subtelomereand telomere in B-cell lines

Chromosomal instability was also analyzed moreprecisely by the interphase FISH method using three sub-telomere probes at the long arm of chromosome 1 (Tel1q), the short arm of chromosome 3 (Tel 3p), and thelong arm of chromosome 7 (Tel 7q), which was per-formed at about every 20 passages. These subtelomereprobes are localized about 200 kilobases from each telo-mere region. This assay is shown schematically in Fig. 3.Some nuclei had multiple copies of subtelomere signalswith two normal centromere signals, which indicates thatthe subtelomere region is translocated to other chromo-somal regions and duplicated. This phenomenon is simi-lar to segmental jumping translocation of chromosomalsegments (SJT) found in chemo- or radiotherapy-relatedsecondary leukemias and atomic bomb radiation- inducedleukemias with a highly complex karyotype, which wefirst reported [26]. This assay may also be able to detectmonosomy, trisomy, and partial deletion. The interphaseFISH method using a subtelomere probe is simple andsuitable for evaluating radiation-induced chromosomal in-stability in the human population. T8-8 from 60Co g-rayirradiation and the T13-1 cell line from 241Am a-ray ir-radiation had more aberrations than the nonexposed T14-6 cell line (Fig. 4). Abnormalities involving subtelomereregions of Tel 1q and Tel 3p were induced at a higherfrequency in cells exposed to 241Am a-rays and 60Cog-rays, respectively. The aberration yields obtained fromthe two different radiation sources, however, did not dif-fer. Monosomy 7 or deletion of the long arm of chromo-some 7 (e7/7qe) and monosomy 5 or deletion of thelong arm of chromosome 5 (e5/5qe) were also found

http://f

Fig. 2. Change of spontaneous chromosome aberration frequencies in two

B-cell lines (T8-8 from g-rayeexposed lymphocytes) at passages 1e9. To-

tal, total percentage of spontaneous chromosome aberrations; 3N, 4N,

polyploidy; ctb, chromatid breaks; ctg, chromatid gaps; csb, chromosome

breaks; cte, chromatid exchange; end, endreduplication; St, structural ab-

errations such as translocation or marker chromosomes, D/R, dicentrics

and rings.

Fig. 1. Change of spontaneous chromosome aberration frequencies in two B-cell lines (T13-1 from a-rayeexposed lymphocytes and T14-1 from non-irra-

diated lymphocytes) at passage 1e5. Total, total percentage of spontaneous chromosome aberrations; 3N, 4N, polyplody; ctb, chromatid breaks; ctg,

chromatid gaps; csb, chromosome breaks; cte, chromatid exchange; end, endoreduplication; St, structural aberrations such as translocation or marker chro-

mosomes; D/R, dicentrics and rings.


more frequently in the T8-8 cell line of the 60Cog-rayeexposed cell line.

Abnormalities in the telomere region were also analyzedby metaphase FISH using a telomere probe. T8-8 from 60Cog-ray exposure had 19.5% of spontaneous chromosome ab-errations at passage 8. Twelve percent of metaphases hadtelomere fusion in passage 8 of the T8-8 cell line, in whichtwo telomere signals were observed in the junction regionfor telomere fusion. T13-1 from 241Am a-ray exposure had3.3% telomere fusion, the value of which was higher thanthat of T14-1 from nonexposed cells (1.9%).

The present study demonstrates that human lymphocyteswith a history of exposure to a- and g-rays show greaterchromosomal instability at several passages after irradia-tion, and also clearly indicates that radiation-induced chro-mosomal instability develops continuously in subtelomeresand telomeres at every passage in descendant cells culturedfrom a single B-lymphocyte after irradiation.

3.4. DNA-PK activities and protein expressions of Ku70and p53

Protein expression level of Ku70 and DNA-PK activities:We observed the doseeresponse relationship of the dicen-tric and ring chromosome yield in eight B-cell lines withand without high genetic instability and five sublinesderived from the T13-1 cell line re-irradiated with 0.5e4Gy of 60Co g-rays. All B cell-lines had higher radiosensi-tivity than the fresh lymphocytes derived from the same do-nor, which were used to establish these B-cell lines.

The protein expression level of Ku70 was observedperiodically in the three cell lines (T14-2, T13-1, and T7-3)by the immunostaining method. The protein level wasincreased at 12e24 hours, after 2 Gy of 60Co g-ray

Table 2

Chromosome instability in 241Am a-raye and 60Co g-rayeirradiated and non-exposed B-cell lines, detected by sequential chromosome and FISH

observations

Passage P1 P4 P10 P20 P27 P38 P46 P56 P86 P90 P96 P100 P117

T14-1 (nonexposed)

Clonal chromosome

aberrations

46,XY 46,XY 46,XY,t

(7;18)(p11;q22)/

idem,add(4)(p13)

Spontaneous chrom.

Aberrations (%)

6.4 5.2 6.0 4.0 5.0 7.0 8.0

FISH:D12Z1;þ12(%): 1.9

WCPY;-Y(%); 4.1 1.7 0

þY(%) 3.4

T13-1(a-ray)

Clonal chromosome

aberrations

46,XY 47,XY,þ12/46,XY

47,XY, del(11)(q24) þ12 47,XY, del(11)(q24) þ12/47

idem, add(13)(p10),add

(15)(p10)

Spontaneous chrom.

aberrations (%)

18.6 15.8 24.0 26.0 58.9 31.0 28.0 24.0

FISH:D12Z1;þ12(%): 1.5 29.2 23.1 5.1

WCPY;-Y(%); 0.38 0.98

þY(%) 0.95 0

T8-1 (g-ray)

Clonal chromosome

aberrations

46,XY 46,XY 46,XY,t(1;11)

(p36;q23)

/near tetraploid

42%

46,XY,t(1;11)

(p36;q23)/

near tetraploid

33.3%

Spontaneous chrom.

aberrations (%)

12.8 17.6 15.0 81.0 15.0 17.0

FISH:D12Z1;þ12(%): 13.1 16.3 22.4

WCPY;-Y(%); 1.37 1.40 8.4 1.0

þY(%) 0.64 1.30 16.1

Fig. 3. Method for evaluating delayed chromosome aberration rates in interphase nucleus by the FISH method. Segmental jumping translocations are de-

tected easily with this method. This method can also detect terminal deletions as well as monosomy. In the interphase nucleus, the FISH method can detect

chromosomal instability involving the subtelomere region.


Fig. 4. Change of the frequencies of chromosome abnormalities involving three subtelomere regions in three B-cell lines (T8-8, T13-1, and T14-6) detected

by interphase FISH. T8-8, T13-1, and T14-1 were derived from g-raye and a-rayeexposed and nonexposed lymphocytes, respectively. FISH analysis was

performed in the interphase nucleus serially from culture passages 1e9 (P1eP9) in the T8-8 cell line, from passages 1e4 (P1eP4) in the T13-1 cell line, and

from passages 1e10 (P1e P10) in the T14-6 cell line. P, passage.


irradiation, but the level decreased in the control B-cell lineT14-2 at 26 hours. On the other hand, the expression levelsof Ku70 protein in both the T7-3 and T13-1 cell lines afterexposure increased at 13e26 hours, at G2 phase, but werelower than that of the T14-2 cell line. This was confirmedby Western blot (Fig. 5).

DNA-PK activity is acquired by binding with the threeproteins of Ku70, Ku80, and DNA-PKcs. The activity levelseems to correlate with the levels of chromosomal instabil-ity. DNA-PK activities were measured by filter-binding as-say. The expression levels of the four B-cell lines derivedfrom a-raye, g-raye, and non-irradiated lymphocytes

Fig. 5. Periodic analysis of Ku70, p53, and p21 protein expression after re-irradi

phocytes (T13-1A’ and T13-1F).

(T13-1, T7-3 and T8-4, and T14-1, respectively) had lowactivity in the non-irradiated cell lines. T13-1 and T8-4 celllines showed an increase to high and medium levels soonafter 2 Gy of 60 Co g-irradiation, respectively, but theT7-3 and T14-1 cell lines showed no increase (Fig. 6).

Protein expressions of p53 and p21: The p53 proteinexpression levels were analyzed by immunofluorescentstaining. T14-1 and T8-2 cell lines derived from non-irradi-ated and g-rayeirradiated lymphocytes, respectively, hadhigher amounts of p53 protein than the T13-1 cell line at3 and 20 hours after 2 Gy of 60 Co g-ray irradiation(Fig. 7). Western blot analysis showed that the amount of

ation of 2 Gy of g-rays in B-cell lines derived from a-rayeirradiated lym-

Fig. 6. Periodic analysis of DNA-PKcs activities after re-irradiation of 2

Gy of g-rays in B-cell lines derived from a-raye and g-rayeirradiated

lymphocytes (T13-1A and T7-3 and T8-4, respectively) and no irradiated

lymphocytes (T14-1).


p53 protein was increased at 18e36 hours, at the G2 phase,but there was no increased p21 expression (Fig. 5). No genemutations were detected in exons 5e8 of the Trp53 gene inthe five cell lines with high chromosome instability and inthe three cell lines from non-irradiated lymphocytes, whichwas detected by the PCR-SSCP method.

3.5. Expressions of telomerase and TRF 1 protein

Telomere regions were frequently associated with de-layed chromosome instability, which indicated that abnor-malities of telomere proteins seemed to be associated with

Fig. 7. p53 protein expression in B-cell lines (T13-1 from a-raye, T8-2 from g-

at 3 and 20 hours after re-irradiation of 2 Gy of 60Co g-rays. FITC signal inten

the development of delayed genetic instability. TRF1 havebeen isolated as binding proteins associated with humanand mammalian telomeres [27e29]. We studied the expressionlevels of telomerase and TRF1 protein. TRF1 protein expres-sion levels were analyzed by immunofluorescent staining24 hours after 60Co g-ray irradiation at a dose rate of300 mGy/min. Both the T8-2 and T14-1 cell lines had a high-er increase of TRF1 protein, but the T13-1 cell line had al-most no increase (Fig. 8). Western blot analysis of TRF1protein in the T13-1 cell line also showed loss of 20- and300-kD bands 24 hours after 2 Gy of 60Co g-ray irradiation,indicating a small amount of TRF1 protein (data not shown).

Amounts of telomerase in T13-1 and its sublines, the T8-4and T14-1 cell lines, at 60 hours after 2 Gy of 60Co g-rayirradiation, which were derived from a-raye, g-raye andnon-irradiated lymphocytes, respectively, were determinedby the in situ TRAP assay. These expression levels werenot different, however, although they were almost the sameas fresh lymphocytes at 60 hours after 2 Gy of 60Co g-rayirradiation, and those were much higher than that of non-irradiated fresh lymphocytes (data not shown). No relation-ship was observed between the amount of telomerase andradiation-induced chromosomal instability.

T13-1 cell line, which showed high chromosome insta-bility, had low amounts of Ku70, p53, and TRF1 proteinsinduced after g-ray irradiation. The expression levels ofthe three proteins had a positive correlation with radia-tion-induced chromosomal instability. The protein resultsof these B-cell lines are summarized in Table 3.

3.6. SCE in irradiated B-cell lines

SCE numbers were not significantly increased by 2 Gy of60Co g-rays (200 mGy/min) in the T13-1 cell line establishedfrom 241Am a-irradiated lymphocytes, two cell lines (T8-1)established from 60Co g-rayeirradiated cells, and the T14-2cell line established from non-irradiated cells. These values,

raye irradiated lymphocytes, and T14-1 from non-irradiated lymphocytes)

sity was analyzed by laser microscope in 1,000 cells.

Fig. 8. TRF1 protein expression in B-cell lines (T13-1 from a-raye, T8-2

from g-raye irradiated lymphocytes, and T14-1 from non-irradiated lym-

phocytes) at 24 hours after re-irradiation of 2 Gy of 60Co g-rays. FITC sig-

nal intensity was analyzed by laser microscope in 1,000 cells.


with and without radiation exposure, were as follows: 8.88 6

2.48 and 8.19 6 4.07 in the T13-1 cell line, 12.7 6 3.09 and12.7 6 4.9 in the T8-2 cell line, and 11.5 6 4.06 and 12.0 6

3.37 in the T14-2 cell line. The T13-1 cell line derived froma-irradiated lymphocytes showed almost the same value asthe donor (7.8), but T8-2 from g-irradiated cells and T14-2from non-irradiated cells showed 12.7 and 11.5, respectively,values higher than in the T13-1 cell line.

4. Discussion

4.1. Delayed chromosome aberrations of B-cell linesand radiation quality

Both 241Am a-rays and 60Co g-rays could induce delayedchromosomal instability. The possibility of LET dependency

Table 3

p53, ku70 , TRF 1 and telomerase expressions in eight B-cell lines

Cell lines

Radiation

sensitivity ** p53b*, *** Ku70a,***

T13-1A (a) þþ þ/� þ/�T13-1F (a) þþ ND ND

T7-3 (g) þ/� þ þT8-2 (g) þ þþ ND

T8-4 (g) þ ND ND

T8-8 (g) þþ þ/� ND

T14-1 (No) þ þþ ND

T14-2 (No) þ/� þ þSublines T13-1A and T13-F are derived from the original T13-1 cell line. Th

lines with high chromosome instability, as shown in Table 1.

The radiation source used to derive each cell line is in parentheses. (No) ind

Levels of protein expression: þ/þþ high response, þ/� low response, e no

Abbreviation: ND, not done.a Analyzed by Western blot method.b Analyzed by immunofluorescent staining and microscopy using software.

* No detectable mutations at exon 5-9 by PCR-SSCP.

** Analyzed chromosome aberration rates after irradiation of 0.5e4 Gy with

*** After irradiation with 2 Gy of 60Co g-rays.

on the delayed chromosome aberrations was also evident inour studies on the HL-60 human leukemia cell line, in whichinterphase FISH analysis revealed higher instability of chro-mosomal segments involving the three subtelomere regions(Tel 1q, Tel 3p, and Tel 7q) on 241Am a-ray exposure than60Co g- ray exposure (data not shown). Similar results wereobserved in transplantation assays, which demonstratedthree- and twofold higher incidences of chromosome insta-bility in the progenies of a-irradiation and 252Cf-neutron irra-diation, respectively, than in x-ray irradiation [30]. Their dataare consistent with an LET-dependent induction of chromo-some instability in the hemopoietic cells.

4.2. Plausible mechanisms for developing delayedchromosome aberrations

Defect functions of telomere and subteleomere: Observa-tion of these B-cell lines showed that a several percentageof the delayed chromosome aberrations occurred at centro-mere, telomere, and subtelomere sites, which are not al-ways involved in chromosome aberrations soon afterexposure. The different distributions of chromosome break-points between fresh lymphocytes just after irradiation andthese B-cell lines from the same donor also confirmed thatthe types of aberrations induced soon after irradiation anddelayed chromosome aberrations were different, anddelayed chromosome aberrations had breakpoints moretelomeric side of each chromosome (data not shown). Cellsharboring telomere fusion, hyperploidy, hypodiploidy, andcentromere spreading seemed to have a defect in telomereor centromere function. Chromosome types in delayedchromosome aberration were different from those thatdeveloped after direct irradiation, suggesting that different

DNA-PK TRF1a, *** TRF1bAmount of

telomerase

activity ***

(in situ TRAP

assay)

þþþ þ/� � þND ND ND ND

þ þ ND þND ND þþ ND

þþ ND ND ND

ND ND ND þþ ND þþ þND þþ ND ND

e five cell lines T13-1, T7-3, T8-2, T8-4, and T8-8 were identified as cell

icates there was no irradiation.

response.

60 Co g-rays.


mechanisms for the induction of these chromosome aberra-tions might be involved.

Telomere loss is a mechanism for chromosome instabil-ity in mouse embryonic stem cells and human cancer cells[31e34]. An increase in the number of subtelomere signalson interphase cells is induced by partial duplication,trisomy, and SJT of the chromosome involving the subtelo-mere region. Recent cytogenetic and DNA sequence analy-ses in another laboratory revealed that pieces of thesubtelomere have changed location and copy numberduring primate evolution [35]. Although the reason whydelayed chromosome aberrations involved in the moresubtelomeric or telomeric regions is not clear, we devel-oped a hypothesis based on the telomere breakageefusioncycle model [36]. Most dicentric chromosomes induced di-rectly by radiation exposure do not have breakpoints attelomere sites. On the other hand, although telomere asso-ciation appearing as a delayed chromosome aberration is ofsimilar shape to the dicentric chromosome, both are totallydifferent. Telomere association has a breakpoint at telomeresites, and the telomere sites of these two types of aberra-tions have quite different functions. On the other hand,about half of the dicentric chromosomes induced by radia-tion are eliminated at every cell division, whereas telomereassociations are not. Recurrent chromosomes may acquiresubtelomere sites at each cell division by the mechanismof telomere capture [37], in which a homologous or hetero-geneous chromosome region located near the telomere,such as a subtelomere site, stabilizes the chromosomeend. The mouse embryonic cells with introduction of dou-ble-strand breaks near a telomere did not undergo sisterchromatid fusion, which followed chromosomal instabilityresulting from breakageefusionebridge cycles involvingthe sister chromatids and rearrangements with other chro-mosomes [34]. These results suggest that telomere-bindingproteins might be associated directly with the developmentof delayed chromosome aberrations through a defect of thecell cycle checkpoint. The T13-1 cell line established from241Am a-ray irradiation had less TRF1 protein and celldivision delay (Tables 1and 3). Ku proteins also associatewith telomeres in mammalian cells and interact with knowntelomere-associated proteins [38,39]. Loss of Ku proteinleads to telomere shortening in mammalian cells [38]. LikeKu protein, DNA-PKcs also localizes to the telomere [38].Taken together, therefore, DNA-PK plays a role in telomeremaintenance that serves to prevent telomereetelomerefusion events.

It remains possible that increased chromosomal instabil-ity also may be caused by the integration of EBV genomesin the B-cell line in some cases. The present results, how-ever, clearly show that B-cell lines established from radia-tion-exposed lymphocytes are significantly more unstablethan those from non-irradiated lymphocytes.

Clastogenic factors contained in culture medium: Theconsequently, persistently expressed cytokines might indi-rectly initiate genetic alterations and chromosome

aberrations [40e42]. Free radicals cause gross cellulardamage, resulting in depletion of nucleotide pools and dis-turbance of sulphur-containig enzymes [43]. It is wellknown that cells exposed extracellularlly to superoxideshow high levels of cytogenetic damage, especially chro-matid-type aberrations [44,45]. The contribution to chro-mosomal instability by the persistent oxidative stress ofa long-term culture medium has been described [46].

Human B-cell lines (T13-1, T8-2, and T14-2) weresubjected to alkaline comet assay to check whether thepassage level of a cell line exerts any influence over tailmoment or radiation-induced genotoxicity [47]. The valuesof tail moment in T13-1 and T8-2 cell lines increased morewith passage levels than in the T14-2 cell line from non-exposed cells. This also indicates that enhanced chromo-some instability in the two cell lines established fromirradiated lymphocytes could be caused by the clastogenicfactor contained in the culture medium [47]. We have alsostudied the clastogenic factors contained in plasma fromhuman lymphocytes after neutron irradiation by alkalinecomet assay to learn about media transfer effects, and wefound that the genotoxic effects lasted for only 96 hourswhen the media were stored at e20�C [48]. This is believedto be mediated by the release of cytokines or other factorsfrom irradiated cells. It still remains to be clarified, how-ever, what kind of clastogenic factors had an effect onDNA or chromosomal damage with such a long incubationtime after irradiation in the present B-cell line experiment.

Cells hit by high LET 241Am a-rays are killed soon afterexposure. Therefore, after exposure, the survivingB-cells are considered to have been untouched by a-raybeams. The B-cell line (T13-1) established from lympho-cytes exposed to 241Am a-rays took longer before establish-ing and had a significantly higher chromosome aberrationrate than the B-cell lines derived from nonexposed lympho-cytes (T14-1 and T14-6). These results suggest that the ini-tial a-ray beameinduced damage not only involves theDNA, but the cell membrane or cytoplasm as well. Theprogeny of non-irradiated bystander cells have been shownto harbor a persistent genomic instability that must resultfrom initial interactions between the irradiated and non-ir-radiated bystander cells [49]. It was reported that the mech-anism of delayed chromosome aberration is related to thatof the bystander effect [49e51], which implies that directradiation-induced DNA breaks per se may not be essentialfor the initiation of the chromosome instability phenotype.

Abnormality of the DNA recombinant protein: As a plau-sible explanation for the induction of the unstable pheno-type, it could be considered that a long time afterexposure, the remaining DNA damage that escaped therepair machinery will be recognized as an unpreparedDNA site, which is called transmission memory [52].Several DNA-binding proteins, such as Ku70, Ku80, andDNA-PKcs, as well as telomere and centromere proteins,would play an important role in this process and bind tothe unprepared double-strand breaks to perform


unscheduled repairs, which induces secondary recombina-tion in a chromosomal region near the primary DNA dam-age, which in turn could induce delayed chromosomeaberrations. The secondary recombination frequently oc-curs in mouseehuman hybrid cell lines incorporated withpart of one human chromosome, which has high chromo-some instability [53], and infrequently in fresh leukemiccells with complex-type translocations, such as t(9;22)[54]. The expression of the Ku70, Ku80, and DNA-PKcsproteins in the B-cell lines (T13-1 from 241Am a-rayeirradiated cells and T8-2 from 60Co g-rayeirradiated cells)were slightly lower than T14-2 from nonexposed cells afterstimulation by irradiation (Table 3). Mechanisms underly-ing higher expression of DNA-PK activity induced by g-ray irradiation, irrespective of low expression of Ku70,might be linked with high chromosome instability in theT13-1 cell line.

Unexpectedly, no increase of SCE was observed in theT13-1 cell line from 241Am a-rayeirradiated cells, in con-trast with the other two cell lines (T8-2 from 60Cog-rays- irradiated cells and T14-2 from non-irradiatedcells). It is well known that homologous recombination(HR) is a mechanism for SCE formation, and the SCE num-ber increases by a-ray irradiation, but not by X- org-ray irradiation [55]. Therefore, these findings imply thepossibility that defective expressions of HR proteins, suchas Rad51, are related with higher chromosomal instabilityin both the T13-1 and T8-2 cell lines.

Altered cellular response: The protein expression of p53was reported as not being associated with delayed chromo-some instability [56], but our study disputes this. T13-1 andT8-8 cell lines established from irradiated lymphocytesexpressed slightly less p53 protein than T14-2 from nonir-radiated lymphocytes (Table 3). Since the studies on cellu-lar response in cells showing delayed chromosomalinstability are quite few, further study will be necessaryto resolve how the cellular responses immediately afterexposure and in delayed chromosome aberrations aredifferent.

4.3. Conclusions

In conclusion, recurrent cytogenetic characteristics inradiation-induced delayed chromosome aberrations weredetected in present study. Multiple mechanisms seem tobe linked to the development of radiation-induced delayedchromosome aberrations. The question of whether theinstability may predispose cells to carcinogenesis stillremains. New concepts regarding delayed chromosomeaberrations will be important to explain the mechanismsunderlying radiation-induced carcinogenesis. It should benoted that the initiation of radiation carcinogenesis maynot be a targeted mutational event, but rather, an indirectprocess that involves a significant fraction of the irradiatedcell population.

Acknowledgments

Irradiation with 241Am a-rays was performed at theRadiation Research Center, Kyoto University. We thankDr Y. Ejima for technical help with a-ray irradiation anddosimetry. We are grateful to Mr. S. Takeoka, Mr. K. Kita-gawa, and S. Suga of the Radiation Research Center of theResearch Institute for Radiation Biology and Medicine,Hiroshima University. We also thank Ms. K. Fujioka andMs. M. Saeda of the Radiation Research Center of theResearch Institute for Radiation Biology and Medicine,Hiroshima University, for excellent technical assistancewith chromosome analysis.

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