Differential Induction of Chromosomal Instability by DNA Strand-Breaking Agents'

ICANCER RESEARCH57. 4048-4056. September 15. 19971 Differential Induction of Chromosomal Instability by DNA Strand-breaking Agents'

Charles L. Limoli,2Mark I. Kaplan, John W. Phillips, Gerald M. Adair, and William F. Morgan

Department of Radiation Oncology [C. L L, M. 1. K., W. F. M.] and Graduate Group in Biophysics (M. I. K.], University of California, San Francisco, California 94143-0750; Life Sciences Dis'ision, Lawrence Berkeley National Laboratory, Berkeley, California 94720; Department of Molecular and Cell Biology. University of Cal,fornia. Berkeley, California 94720 (J. W. P.]: and Unis'ersiO' of Texas M. D. Anderson Cancer Center, Science Park Research Division, Smiths'ille, Texas 78957 (G. M. A.]

ABSTRACT chromosomal change. Indeed, the preponderance of chromosome re arrangements identified in neoplasms serves to emphasize the impor To investigate the role of DNA strand breakage as the molecular lesion tance of studying processes contributing to the formation of chromo responsible for initiating genomic instability, five different strand-break somal rearrangements (5, 6). ing agents, bleomycin, neocarzinostatin, hydrogen peroxide, restriction endonucleases, and ionizing radiation, were examined for their capacity to Cells exposed to ionizing radiation can become genomically unsta induce delayed chromosomal instability. These studies used GM1O115 ble as manifested by dynamic chromosomal destabilization. This human-hamster hybrid cells, which contain one copy of human chromo genomic instability can be observed cytogenetically as chromosome some 4 in a background of 20â€”24hamsterchromosomes. Chromosomal aberrations and rearrangements multiple generations after irradiation instability was investigated using fluorescence in situ hybridization to (7â€”12).The consequences of damage-induced genomic instability visualize chromosomal rearrangements involving the human chromo include delayed mutation, cell fusion, transformation, delayed repro some. Rearrangements are detected multiple generations after treatment, ductive cell death, and gene amplification (reviewed in Ref. 4). The In clonal populations derived from single progenitor cells surviving treat majority of DNA-damaging agents produces genetic lesions that are ment of the specified DNA-damaging agents. Clastogenic and cytotoxic activities of all agents were tested by examining chromosome aberration rapidly repaired and seldomly persist for more than 4â€”6h (13, 14). yields in first-division metaphases and by clonogenic survival assays. This makes it difficult to reconcile how a transient exposure to a Analysis of over 250 individual clones representing over 50,000 met noxious agent can result in such prolonged and persistent destabili aphases demonstrates that when compared at comparable levels of cell zation of the genome. Nonetheless, exposure to ionizing radiation can kill, ionizing radiation, bleomycin, and neocarzinostatin are equally effec initiate the processes associated with genomic instability, which can tive at eliciting delayed genomic instability. These observations document, ultimately contribute toward an increased rate of acquisition of alter for the first time, the persistent destabilization of chromosomes following ations in the genome (4). chemical treatment. In contrast, the analysis of nearly 300 clones and The capacity of ionizing radiation to compromise genomic stability 60,000metaphases,involvingtreatmentwith four different restriction in the progeny of GMIO1 15 cells surviving acute exposure to ionizing endonucleases and/or hydrogen peroxide, did not show any delayed chro mosomal instability. These data indicate that DNA strand breakageper se radiation provides a unique model system with which to study the does not necessarily lead to chromosomal instability but that the complex initiation and progression of genome destabilization (9). Emerging ity or quality of DNA strand breaks are important in initiating this views concerning the underlying mechanisms behind genomic insta phenotype. bility must identify the critical intracellular target(s) and potential molecular interactions capable of initiating this process (4, 15). The INTRODUCTION molecular mechanisms initiating genomic instability must account for the long-lived and dynamic nature of chromosomal destabilization. The propensity of cancer cells to show multiple mutations and We report an investigation involving five agents known to produce karyotypic abnormalities underscores the concept that multistep car specific types of DNA strand breaks, with different chemical termini, cinogenesis progresses by the accumulation of discrete genetic alter i.e., bleomycin, neocarzinostatin, hydrogen peroxide, restriction en ations. Central to understanding the cause and effect relationships donucleases, and ionizing radiation, and how these modulate the between the many end points associated with neoplasia is a knowl induction of delayed genomic instability. The data presented entail the edge of how this genetic change occurs and what proliferative advan analysis of over 550 individual clones representing the scoring and tages are bestowed on the cell. Accumulating evidence suggests that classification of over 100,000 metaphases. The variable induction of genomic instability may provide the driving force behind the genetic delayed genomic instability found after treatment of cells with each of plasticity characteristic of cancer cells (1â€”3).Genomic instability is an these five agents is discussed in regard to the specific types of DNA all-embracing term that includes such changes as gene mutation, gene damage produced. amplification, chromosomal destabilization, and cellular transforma tion (4). Substantial effort aimed at studying cancer progression has focused on characterizing specific treatments capable of inducing the MATERIALS AND METHODS same end points observed in cancer cells and on developing systems capable of accurately measuring the resultant changes. Successful Cell Culture application of these approaches has established that specific genetic Two genetically distinct subclones of the human/hamster hybrid cell line lesions can lead to mutations, many of which result from gross GMlOllS were used for these studies. GMIOI 15cells contain a single copy of human chromosome 4 in a background of 20â€”24 hamster chromosomes. Received 5/5/97; accepted 7/I 8/97. GMIO1l5â€•@cellscontain two copies of the APRT3 gene, and GMl0ll5@'Â° The costs of publication of this article were defrayed in part by the payment of page cells are hemizygous at the APRT locus. Both cell lines were maintained in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DMEM supplemented with 10% fetal bovine serum, 2 mML-glutamine, 100 I This work was supported by the Office of Health and Environmental Research, units of penicillin, 100 mg/mI of streptomycin, and 0.2 mist L-prohne. Cells United States Department of Energy, Contract 4459-01 1542; NIH Grant GM 541 89 (to were maintained at 34Â°Cinhumidified incubators containing 5% CO2in air. w. F.M.); NIH NationalResearchServiceAward5-T32-ES07016fromthe National Institute of Environmental Health Sciences (to C. L. L. and J. W. P.); and USPHS Grant

CA-2871 I from the National Cancer Institute (to G. M. A.). 3 The abbreviations used are: APRT, adenine phosphoribosyltransferase; RE, restric 2 To whom requests for reprints should be addressed. at Department of Radiation tion enzyme; DSB, double-strand break; SSB, single-strand break; BLM, bleomycin; Oncology,Box0750.UniversityofCalifornia.SanFrancisco,CA94143-075.Phone: NCS, neocarzinostatin; FISH, fluorescence in situ hybridization; D37, the drug concen (415) 476-2793; Fax: (415) 476-1544; E-mail: [email protected]. tration needed to reduce the fraction of surviving cells from I to I/c. 4048

Isolation and Characterization of GM1O11Sâ€•Â°Cells Hydrogen Peroxide. GMlOll5@ cells (I X l0@cells/mI, 10 ml total) were incubated with the desired concentration of H,O, (Sigma Chemical Co.) Spontaneous APRr'Â° (hemizygous) GMlOl 15 sublines, which have lost for 30 mm at 34Â°C.Alltubes were maintained at equal cell densities during one copy of the Chinese hamster APRTgene, were selected on the basis of their drug treatment to minimize intracellular variations in H2O2 concentration due resistance to intermediate levels of 8-aza-adenine (16â€”18).GMlOll5@ cells to catabolic consumption by catalase. For simultaneous treatment of cells with were subcloned to derive a series of independent, clonally derived cell popu H2O2and the RE StuI, cells were first electroporatedwith Stul and then lations. Expanded clonal populations were then plated at lO@cellsper 100-mm immediately diluted into prewarmed medium containing the desired concen tissue culture dish with selective medium containing 8 p@g/mlof8-aza-adenine. tration of H2O,. The mediumwas replacedwith fresh selection mediumevery 4 days. Single BLM. Twenty-five-cm2flasks containing2â€”3X10â€•GMlOll5@@cells clones surviving selection were then picked, grown to mass culture, and were incubated with the desired concentration of BLM ( I .0â€”300 @xg/ml; characterized by Southern blot analysis to determine whether they were het Blenoxane Squibb) for 4 h at 34Â°C. erozygous or hemizygous for the APRT locus. NCS. NCS was a generous gift from Drs. Gene Joiner and L. Gayle Littlefield at the Oak Ridge National Laboratory (Oak Ridge, TN). One million Restriction Enzyme Treatment of Cells and Selection of APRT GMlOl l5@@(1 X lO@cells/mI)were incubated with the desired concentration Mutants of NCS (0. 1â€”10@xgIml)for I h at 34Â°C. Immediately following the appropriate treatment, cells were rinsed to re The hemizygous GMlOl l5@'Â°cellswere generated specifically for the move residual drug and resuspended in fresh complete medium before plating. detection of APRT mutants following treatment with REs. Restriction enzyme Those cells not destined for experiments involving the isolation and identifi cleavage and subsequent misrepair at the unique recognition site within the cation of delayed genomic instability were replated for the analysis of first endogenous APRTgene generate APRTmutations and provide evidence that an division metaphases. enzymatically active endonuclease entered and produced DNA DSBs within a given cell (19). Mutations were induced by treatment of these APRr'Â° cells Cell Survival and Colony Isolation with one of four restriction enzymes obtained from Boehringer Mannheim @ (Indianapolis, IN): BsaHI (GPu CGPyC; 100 units/electroporation, 10 units/ Immediately following irradiation or chemical treatment, cells were diluted @ @xl);PvuII (CAG CTG; 25 units/electroporation, 10 units/xl); StuI and plated in triplicate into 100-mm dishes containing 10 ml of medium to @ (AGG CD'; 10 and 15 units/electroporation,10 units/pi); or EcoRV determine the surviving fraction. The number of cells plated was based on the @ (GAT ATC; 300 and 450 units/electroporation, 50 units/pA). Each RE expected level of kill for a particular agent and on limiting the number of cleaves only once within one of the five exons of the endogenous APRT gene colonies per plate (â€”20) to minimize the chances of selecting more than one and was introduced separately by electroporation into cells as described individual clone during colony isolation. After 2â€”3weeks, distinct and well previously (19). isolated colonies of 100 cells had formed. Some of these colonies were APRr'Â° cells were selected by plating 2â€”3X lO@RE-treatedcells into picked at random from each of the plates using sterile Dacron swabs dipped in 100-mm dishes with 10 ml of selective medium containing 80 @xg/mlof trypsin and expanded to mass culture (â€”1â€”3X106cells) in 25-cm2flasks. The 8-aza-adenine. Two to 3 weeks of growth were generally required before APRT remaining colonies were stained with I % crystal violet in 25% ethanol and mutant colonies reached a size sufficient for subsequent counting, isolation, counted. Cell survival was determined as the number of colonies picked plus and expansion. Isolation of mutant colonies and subsequent characterization of the number of colonies stained divided by the number of cells plated times the the APRT mutation at the molecular level allow us to determine whether an plating efficiency. Those colonies expanded to mass culture were prepared for endonuclease entered the cell and induced the observed mutation. analysis of potential chromosomal instability as described below.

Analysis of APRT Mutations Collection of Metaphase Cells

Genomic DNA was isolated and purified from expanded APRT mutant Metaphase cells derived from clonally expanded cell populations were clones by a â€œsaltingoutâ€•procedure(20). PCR amplification across specific collected by mitotic shake off following a 2â€”3-hincubation in the presence of regions of the APRTgene was carried out by selecting primer pairs that flanked the mitotic spindle inhibitorColcemid (final concentration, 2 X lO@ M). Cells specific exons containing the unique restriction site. These regions of the APRT were then swollen in hypotonic 0.075 M KCI solution for 15 mm at 37Â°C, gene were amplified by PCR in a total volume of 100 @xl,usinga standard dehydrated in 100% methanol, and fixed in a 3: 1 mixture of methanol:acetic 40-cycle amplification protocol and reagents from the GeneAmp DNA ampli acid. Mitotic cells were dropped onto precleaned glass microscope slides and fication kit and 0.5 units of AmpliTaq DNA polymerase (Perkin-Elmer, allowed to air dry at least 2 days at room temperature before storage at â€”20Â°C. Norwalk, CT). Primers were 18 mers and used at SOpmol/reaction in con junction with 0.5 @xgofgenomic DNA. Amplified regions were analyzed for FISH mutations by restriction fragment length analysis; 20-@xlaliquots of PCR product were digested with the same restriction enzyme used to induce the Posttreatment analysis of chromosome aberrations at the first metaphase and APRT mutationand resolved via 4% agarosegel electrophoresis.RE-induced of delayed chromosomal instability used FISH of a labeled probe to the human mutations by definition lose the RE site and are refractory to subsequent chromosome in the hybrid cells. The labeling of this pBluescript vector-based enzyme cleavage (19). library of human chromosome 4-specific DNA sequences (pBS4) has been described (9). Analysis of Delayed Reproductive Cell Death in Restriction Slides with metaphase spreads were washed in two changes of 2X SSC (0.3 Enzyme-treated Cells M NaCI and 0.03 M sodium citrate, pH 7) for I 5 mm, then dehydrated in 70, 90, and 100%ethanol for 2 mm each at ambienttemperature.Afterdrying, To ascertain whether clones determined above to be RE-induced or non chromosomes were denatured for 30 s at 80Â°Cin 70% formamide and 2 x SSC RE-induced APRT mutants showed any differences in delayed reproductive and then dehydrated again in the ethanol series described above. After drying, cell death, clonogenic assays were performed to measure plating efficiency. 35 pA of hybridization mix (70% formamide, 15% dextran sulfate, 2 XSSC, Colony-forming assays were performed on 23 individual BsaHI enzyme and SO ng labeled probe) were applied to each slide, which was then covered induced APRT mutants and 10 spontaneous APRT mutants by plating 100 with a glass coverslip and sealed with rubber cement. Slides were incubated a cells/lOO-mmdish in triplicate. minimum of 2 days in a humidified incubator at 37Â°C. All remaining steps were done at ambient temperature. Slides were washed Irradiation and Chemical Treatment of Cells in three changes of phosphate-buffered detergent, once briefly and twice for 5 mm each. For detection of the biotinylated probe, slides were incubated with X-Irradlation. Log-phase cells were exposed to 10 Gy of X-rays at 250 FITC-avidin (Oncor) for 15mm while covered with plastic coverslips (Oncor). cGy/min at ambient temperature using a Phillips RT250 X-ray machine Slides were washed again in three changes of PBS, and the fluorescent signal (250-kV peak, 15 mA; half-value layer, 1.0 mm copper). was amplified via analogous incubation using an anti-avidin antibody (Oncor) 4049

followed by a repeat incubation with the FITC-avidin. After a final rinse and two 5-mm washes in phosphate-buffered detergent, propidium iodide in An tifade (Oncor) was added to slides, which were then covered with glass @,a. ,@â€”10.1 coverslips. ,@st â€”8.0 @ Cytogenetic Analysis $@ a.@ â€”7.1 Visualization of metaphase chromosomes following FISH was accom plished using a Zeiss Axioskop microscope equipped with a dual-band pass FITCITexasRedfilterset. Withthissystemandthecombinationof fluorescent dyes used, the background of hamster chromosomes appears red (propidium iodide emission), whereas the biotinylated probe hybridized to the human chromosome 4 target appears yellow-green (fluorescein emission). Kodak 400 12 34 5 ASA slide film was used for all fluorescence photography,and no digital Fig. I. Southern blot hybridization analysis of the three APRr'Â° hemizygous sublines processing was used to modify any of the presented images. that were derived from the APRr'@ GMlOl 15 cell line. EcoPJ/BclI double digests of Genomic instability for the purposes of this report is defined as any clone genomic DNAs, hybridized with a 3.9-kb BamHI fragment Chinese hamster APRT probe derived from a single cell that shows at least three distinct metaphase sub are shown. Lane 1, parental GMlOl 15 cell line; Lanes 2â€”4,threeindependently derived populations involving rearrangements of human chromosome 4 (9). Analysis APRTâ€•Â°hemizygous sublines; Lane 5, wild-type CHO cells. of chromosomal instability entails scoring 200 individual metaphases from each clone. Only those rearrangements involving human chromosome 4 were scored and categorized as described by Tucker et al. (21). chromosomes; (b) an 8.0-kb EcoRI-Bc!I fragment present only on the 74chromosome;and(c)a 10.1-kbEcoR!-Bc!Ifragmentpresentonly RESULTS on the Z7 chromosome. Similar analysis performed on three mdc pendently derived GM1O1 15 APRTmutants (Fig. 1, Lanes 2â€”4)show Ionizing Radiation Treatment. To establish the frequency with a less intense (single copy) 7.1-kb EcoRl fragment and loss of the which ionizing radiation induces genomic instability, as measured by 8.0-kb EcoRI-Bc!! fragment, indicating that these clones, like other delayed chromosomal destabilization, APRT@'@ and APRT@'Â°cells spontaneous deletion events characterized in CHO strains, have un were exposed to 10 Gy of X-rays. Table 1 shows the results of over dergone complete loss of the CHO Z4 APRT allele (25, 26). Fig. 1, 10 independent experiments where 152 individual clones were iso Lane 5 shows wild-type CHO cells. The APRT@'Â°subline shown in lated from single progenitor cells surviving X-ray exposure. Exami Fig. 1, Lane 4 (subclone D2) was made specifically to monitor nation of more than 30,000 metaphases indicates 10 Gy of X-rays misrepair of RE-induced DSBs and was used in all subsequent cx induce genomic instability in 33% of the clones analyzed. Ten Gy of periments using REs. X-rays induced chromosomal instability in both APRr'@ and RE Treatment. Using REconcentrationsdeterminedpreviouslyto APRT@'@'Â°OMlOl15 cells at a similar frequency. maximize the yield of APRT mutations (19), clonogenic survival for Generation and Analysis ofAPRTâ€•Â°GM1O115 Sublines. Hem all enzymes used was reduced to 10â€”15%.Metaphase chromosomes izygous APRr'Â° sublines of GMlOl 15 CHO cells were generated were prepared from cells treated with RE at the first mitosis after specifically for the experiments involving restriction enzymes. Clones treatment to verify the clastogenic properties of REs in cells. Four selected under intermediate levels of 8-aza-adenine were expanded hundred metaphases examined from two independent experiments in and analyzed by Southern blot analysis (Fig. 1). CHO cells contain which cells were electroporated with equivalent volumes of RE stor two copies of the APRT gene, one located on chromosome 14 and the age buffer without enzyme did not show aberrations. Analysis of 200 other on the Z7 chromosome (22, 23). The GMlOl 15 strain is het metaphases from cells treated with 100 units of BsaHI or 25 units of erozygous for a Bc!! RFLP involving the presence or absence of a Bc!! PvuI! revealed that 62 and 71%, respectively, showed rearrangements site â€œ6.8kb downstream of the APRT gene (24). EcoRl/BclI double involving human chromosome 4. digests of genomic DNA from APRr'@ GMlOl 15 cells (Fig. I , Lane APRT mutant colonies were picked and clonally expanded for both 1) show three bands that hybridize to a 3.9-kb BamH! fragment APRT preparation of metaphase chromosomes and purification of genomic probe:(a)adark(dual-copy)7.1-kbEcoRIfragmentpresentonbothDNA. Using PCR amplification of the region of the APRT gene that surrounded the RE recognition sequence of interest and recleavage Table I Delayed chromosomal instability induced by 10 Gy of ionizing radiation with the same RE that was used in the mutagenesis experiments, we GMlOll5â€•@ cells GMl0ll5@'Â°cells were able to categorize the mutants according to whether they con tamed a change at the recognition sequence for that particular enzyme No. of of metaphase aberrant of metaphase aberrant clonesNo.metaphases'Â°691â€”20â€”361â€”27â€”8221â€”297â€”10083â€”40â€”633â€”412â€”38123â€”485â€”10025â€”73â€”955â€”719â€”7155â€”710098â€”Il84-100subpopulationsa% metaphasesâ€•No.5ub@pulationsa% (Table 2). Fig. 2 shows a typical analysis of 11 APRT mutants isolated after Stul treatment. Fig. 2, Lanes 3â€”5show mutations that did not involve the recognition sequence and are referred to as non-RE induced mutants. Fig. 2, Lanes 6â€”12show mutations that did involve the recognition sequence and are now refractory to enzyme cleavage. These we classified as RE-induced mutants. They typically comigrate with the uncut control (Fig. 2, Lane 2) unless they have undergone recombination resulting in large insertions or deletion at the restriction site (Fig. 2, Lanes 6 and 13). Those mutants that were not amplified 51â€”223â€”5I3I527 0â€”3.5 3â€”4 by PCR using primers flanking the recognition sequence, but could be 80 amplified at other regions of the APRT gene, were also considered 3.5 RE-induced mutants (data not shown). Past work has established that 99-100 these mutants contain complex rearrangements involving the APRT a Number of different abnormal metaphase subpopulations showing distinct types of chromosome aberrations. locus (19). Molecular analysis of 233 APRT mutants indicates that 25 b Percentage of metaphases scored showing chromosomal aberrations. of 40 (62%), 12 of 83 (14%), 25 of 36 (69%), and 61 of 74 (82%) of 4050

INDUCTION OF DELAYED GENOMIC INSTABILITY

REsRestriction Table2 IntluctionofAPRT mutations aad delayed chromosomal rearrangements by of clones of metaphase aberrant enzymeEnzymemutationsBsaHI25 concentrationNo. analyzedNo. subpopulationsâ€•% metaphasesâ€•Enzyme-induced units24 I I 100 Yes 14 1â€”2 0-2.5 No NoPvuII25 I1â€”2 I0â€”2.5 100Yes unitsI I 1 1 100 Yes 68 1â€”2 0â€”5 No 2 2 8â€”10 No NoEcoRV300 I1â€”2 I0â€”10 100Yes units 5 â€”2 9 1 0 No 450 unitsI 10 1â€”2 0â€”2.5 Yes NoStuI10 7I 10-3 0â€”0.5Yes units 1 â€”2 2 1 23â€”57 Yes 5 1â€”2 100 Yes 8 1â€”2 0â€”2.5 No 15 units3 23 1 0-0.5 Yes 51 10â€”2 0Yes No

a Number of different abnormal metaphase subpopulations showing distinct types of chromosome aberrations.

b Percentage of metaphases scored showing chromosomal aberrations.

the clones exposed to BsaHI, PvuII, EcoRV, or Sw!, respectively, had delayed reproductive cell death. The results of the clonogenic assays changes at their respective recognition sites (Table 2). Collectively, used to measure plating efficiencies of each clone are shown in Table 123 (53%) of the mutants were RE-induced. 3. A total of 23 RE-induced APRT mutants (treated with 100 units of For each of the APRT mutants isolated and characterized above, a BsaHI) had a mean plating efficiency of 73 Â±4.8 (Â±SE), whereas a subset of the clonal population was set aside for the preparation of total of 10 non-RE-induced APRT mutants had a mean plating effi metaphase chromosomes. None of the 233 mutant clones analyzed ciency of 73 Â±4.0 (Â±SE). Statistical analyses (ANOVA and Stu demonstrated chromosomal instability, despite analysis of over dent's t test) performed between the data sets of RE-induced and 46,000 metaphases from the RE-induced and non-RE-induced groups non-RE-induced APRT mutants shown in Table 3 were not found to (Table 2). be significantly different. Hating Efficiency in RE-treated Cells. Having determined the Hydrogen Peroxide Treatment. To determine whether H202 was nature of the APRT mutation in a large number of RE-treated clones, capable of eliciting delayed genomic instability, cells were exposed to we sought to determine whether clones known to be either RE a range of concentrations (1.0â€”300 .LM)for 30 mm at 34Â°C.The induced or non-RE-induced APRT mutants showed any differences in survival data for H2O2 exposure are shown in Fig. 3. As before, metaphase chromosomes prepared at the first mitosis were examined I 2 3 4 5 6 7 8 9101112 for aberrations, and after scoring 200 metaphases derived from pop -. A-'@ ulations of cells exposed to H2O2, no chromosome aberrations were @ â€˜.@ detected. Clones isolated from H2O2 concentrations that reduced survival by 2â€”3logs were expanded for analysis of genomic instabil ity. Of the 18, 7, and 6 clones isolated after 100, 200, and 300 @LM 772â€” 661 â€” H202 treatment, respectively, none exhibited delayed chromosomal instability (Table 4). RE and Hydrogen Peroxide Treatment. The capacity to produce RE-induced DSBs in the nucleus and damage other cellular targets with H202 provided an attractive method of modeling the action of ionizing radiation. Consequently, we investigated whether combined exposure to RE and H202 could lead to chromosomal instability in GMl0115@Â° cells. Of the 35 APRT mutant clones analyzed, 24 showed RE-induced mutations, and the remaining I 1 were non-RE induced mutants. None were found to show delayed chromosomal instability (Table 4). BLM Treatment. The ability of BLM to induce genomic instabil ity was investigated by exposing cells to 1.0â€”250p.g/ml BLM for 4 h at 34Â°C.The resulting cell kill is shown in Fig. 4. To further establish the activity of the drug, metaphase chromosomes were prepared at the first mitosis following drug exposure. Two hundred metaphases were 111â€” examined at the first mitosis where 25, 29, 43, and 51% contained multiple complex aberrations involving human chromosome 4, fol Fig. 2. StuI fragments resolved on a 4% agarose gel. Lane 1. 4Xl74 HaeIII-digested lowing treatment with 100, 150, 200, and 250 @g/mlBLM, respec DNA; Lane 2, undigested PCR-amplified StuI fragment; Lanes 3â€”5,mutations not involving the RE recognition site. referred to as non-RE-induced mutants; Lanes 6â€”12. lively. mutations that did involve the RE recognition sequence. referred to as RE-induced At various drug concentrations, survivors were isolated, expanded, mutants. RE-induced mutants shown typically comigrate with the uncut control (Lane 2), and analyzed for genomic instability. Table 5 lists data for 62 clones unless illegitimate recombination of DNA ends results in large insertions (Lanes 6 and 11) or large deletions (none shown). analyzed that survived exposure to 100â€”250p.g/ml BLM. At drug 4051

mutantsRE-inducedÂ° Table 3Plating efficiencies of RE-mduced and non-RE-induced APRT

APRT(%)2Al mutantsPlating efficiency' (%)RE-inducedâ€•APRT mutantsPlating efficiency' (%)Non@RE@inducedbAPRT mutantsPlating efficiency'

3Al 85Â±2 6B2 57Â±8 lB 87Â±2 5Al 73Â±8 2C1 38Â±6 1C 69Â±8 2A2 70Â±6 3Cl 72Â±7 ID 67Â±5 3A2 84Â±12 4C1 92Â±20 lE 79Â±8 4A2 62 Â±9 5Cl 98 Â±3 IF 67 Â±II 5A2 32 Â±4 2C2 74 Â±12 IG 82 Â±13 6A2 62 Â±3 3C2 86 Â±5 lH 68 Â±8 2Bl 102 Â±25 4C2 68 Â±I II 71 Â±8 5Bl 103Â±3 5C2 71Â±9 Ii64Â±6 76Â±2 6BI 95Â±8 6C272Â±4 73Â±16IA 3B261Â±2 87Â±15B2

a APRT mutants derived from cells treated with 100 units of BsaHI.

b APRT. mutants derived from cells treated with BsaHI storage buffer alone.

C Percentage of average plating efficiency with SD.

(yellow) against a background of hamster chromosomes (red). Fig. 5, bâ€”frepresents the types of rearrangements involving the human chromosome that can be encountered in a single unstable clone; the multiple complex rearrangements involving various portions of hu man chromosome 4 demonstrate the dramatic and dynamic nature of

C the end point measured. 0 NCS Treatment. To analyze the capacity of NCS to induce U genomic instability, cells were exposed to 0.1â€”10@.&g/mlNCSfor 1 h U- at 34Â°C.The cell survival curve is shown in Fig. 6. As with BLM, metaphase chromosomes were isolated at the first mitosis to further C confirm the intracellular activity of the drug. Sixteen and 40% of the 200 metaphase chromosomes examined after exposure to I.0 and 3.0 Cl) @.tg/mlNCS, respectively, showed multiple complex aberrations. Cytogenetic data gathered from clones surviving NCS treatment are shown in Table 6. Collectively, 40 clones isolated after 0.5 log kill were analyzed. Table 6 shows that a total of 0 of 14 (0%), 3 of 14 (2 1%), and 5 of 12 (42%) of the clones analyzed after exposure to 1, 3, or 10 @tg/mlof NCS, respectively, were found to be genomically unstable. 0 50 100 150 200 250 300 H202 Concentration (jiM)

Fig. 3. Cell survival following a 20-mm exposure to micromolar levels of hydrogen 100@i:.E::.:@:EiE:.:..::::::::::::.:@:::::::::::::::@:10 peroxide at 34Â°C.All data are fitted with a least mean square straight line of the form y = ce â€œFrom the slope m of this line, [email protected] over the range of exponential cell kill (30â€”[email protected],O2),was calculated to be 25 ,.LM.

TabletheH2O2 4 Delayed chromosomal instability analvzedfollowing exposu to 11202 or combination of H2O2 and the RE Stul (15 units)re concentrationNo.of clones No.of metaphase % aberrant (p@M)mutations100 analyzed subpopulationsâ€• metaphasesâ€•Enzyme-induced 0â€”4.5NA'200 18 1â€”2 0-5NA300 7 1-2 0â€”12.5NA30 6 1â€”2 140â€”2.5Yes2 1â€”2 -F 0-2No60 1â€”2 100â€”4.5Yes9 1â€”2 1â€”2 0â€”1.5No I aNumberofdifferentabnormalmetaphasesubpopulationsshowingdistincttypesof :@I@ !T@:@ chromosome aberrations. S Percentage of metaphases scored showing chromosomal aberrations.

( NA, not applicable.

0.@@@.(@1 concentrations of 100, 150, 200, and 250 @g/ml,4 of 19 (2 1%), 2 of o 50 100 150 200 250 8 (25%), 9 of 16 (56%), and I I of 19 (58%) of the clones, respec tively, were found to be genomically unstable. Fig. 5 depicts examples Bleomycm Concentration (pg/mi) of two clones isolated after exposure to 100 @.tg/mlBLM,one genomi Fig. 4. Cell survival following a 4.-h exposure to BLM at 34Â°C.All data are fitted with cally stable clone (Fig. 5a) and one genomically unstable clone (Fig. a leastmeansquarestraightlineoftheformy = cemÂ°.Fromtheslopemofthisline,D,7 5, bâ€”f).Fig.5a shows the normal unrearranged human chromosome 4 underthedescribedconditionswascalculatedtobe 35 @zg/ml. 4052

BUdBleomycinTable 5 Delayed chromosomal instability induced by colony size and a persistent reduction in plating efficiency (heritable No. of clones No. of metaphase % aberrant lethal mutations), giant cell formation, cell fusion, lowered cell at concentrationmetaphasesâ€•100 (@xgIml) analyzed subpopulationsâ€• tachment ability, delayed mutation, clonal heterogeneity, transforma 150â€”2I 1â€”2 tion, and delayed chromosomal instability (reviewed in Ref. 4). Per 31 4 35I 8 haps the most dramatic of these biological end points is clonal 100I 13 chromosomal destabilization manifesting several generations after 87150 >16 irradiation as visualized by FISH. Ten Gy of X-rays reduce clono 60â€”22 1â€”2 2.5200 3 genic survival by 2â€”3logs and elicit delayed chromosomal instability 0â€”567 1â€”2 in 33% of all clones examined (Table I). Table I shows that nearly 0â€”6I 3-4 one-half of the genomically unstable APRr'@ GMIOI 15 clones 3.5I 5 1001 5 could be considered very unstable; 15% had five or more populations 18250 7 of chromosome aberrations, whereas nine clones showing over 8 70â€”1I 1 1009 1 classes of aberrations were extremely unstable, having markedly 0â€”61 3â€”4 complex rearrangements in nearly all metaphases examined. Of the 11 15.41 6 APRr'Â° clones examined, 6 were unstable, thereby substantiating 56a 8 ofchromosomeNumberofdifferentabnormalmetaphasesubpopulationsshowingdistincttypes that genetic modification of the APRT locus does not preclude the aberrations.b manifestation of delayed chromosomal instability. Percentage of metaphases scored showing chromosomal aberrations. Cellular exposure to ionizing radiation results in a variety of di rectly and indirectly induced DNA lesions, including DNA base DISCUSSION alterations, DNA-DNA and DNA-protein cross-links, and SSBs and DSBs (13). The random nature and variety of lesions inherent to Cells surviving exposure to ionizing radiation demonstrate several radiation complicate elucidation of the primary target and initiating end points collectively termed genomic instability. These include lesion responsible for inducing genomic instability. The effectiveness delayed reproductive cell death, which is revealed by variability in with which ionizing radiation induces delayed genomic instability and

Fig. 5. Examples of chromosome rearrangements foundinonenormalandoneunstableclone,isolatedfrom individual progenitor cells surviving a 4-h exposure at 34Â°Cto 100 @xg/mlofbleomycin. a, normal metaphase showing nonrearranged human chromosome 4. bâ€”d,se rica of photos showing the types of complex rearrange ments involving human chromosome 4 that can be en countered in just one unstable clone.

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:::@EEEEEE@=@::EEE;@EEEEE@E:â€”â€”:-@@::::::10 which produces global cellular damage indiscriminately by virtue of the same reactive species (33â€”35).Cells exposed to H2O2 did not show chromosome aberrations at the first mitosis after treatment, despite the production of prodigious quantities of DNA SSBs and base damages (35). Treatment of cells with micromolar levels of H202 at

0 34Â°Cresults in substantial cell kill (Fig. 3), and despite isolating

C, clones surviving greater than 2-logs kill, none of the 3 1 clones ; analyzed demonstrated chromosomal instability (Table 4). This mdi 1 cates that the action of reactive H2O2-denved hydroxyl radicals throughout both nuclear and nonnuclear intracellular compartments .@ was insufficient to elicit genomic instability, or at a minimum, H2O2 induces this phenotype far less frequently than other treatments pro @iIJ ducing equivalent levels of cell kill. 01:____@L@1â€”----i@@ii:@_@r@i:@E@Ã§-@ â€˜ We expanded this study by including a combination of RE and H,02 treatments to determine whether they may accomplish to gether what neither could do alone. Our rationale was that the 0.01 -@ addition of H2O2 might damage a critical nonnuclear target, which 0 2 4 6 8 10 in conjunction with RE-induced DSBs might elicit genomic insta bility. Alternatively, the addition of H2O2 could generate hydroxyl NCS Concentration (jig/mi) radicals that would produce additional damage at the site of re Fig. 6. Cell survival following a I-h exposure to NCS at 34Â°C.Alldata are fitted with striction cleavage, thereby generating DSBs more similar to those a least mean square straight line of the form v = ce m* From the slope m of this line. D17 produced by ionizing radiation. These premises were not borne out under the described conditions was calculated to be I.4 @sg/ml. experimentally, because the dual action of each agent was again unable to induce any observable genomic instability (Table 4). the preponderance ofevidence implicating DSBs as the critical lesions This dual treatment required the use of H,O, levels lower than responsible for radiation-induced chromosome damage (27), mu treatment with H,O2 alone; otherwise, cytotoxicity was too pro tagenesis (28), gene amplification (29), and cell kill (30) suggest that nounced, which reduced the yield of APRT mutants to impracti the DNA DSB may be the critical lesion responsible for genomic cally low levels. Nonetheless, the fact that we did observe RE instability. To understand the nature of the molecular change that induced mutations argues against the possibility that the RE was sends cells down the pathway of genomic instability, agents known to inactivated by the oxidizing activity of H2O2. produce DNA DSBs with different end structures were investigated. The inability to induce genomic instability with RE and/or H202, REs produce DSBs of known structure having 5'-phosphate and compared to the high frequency with which ionizing radiation was 3'-hydroxyl termini at defined sequences in the genome in the absence able to induce this phenomenon, suggested a fundamental difference of any other DNA lesion. APRT4@1'0hemizygoussublines of GM1OI 15 in the types and/or locations of the lesions produced. To model more cells were constructed specifically for this study to provide the ap faithfully the types and qualities of DSBs produced by radiation, we propriate genetic background to identify cellular cleavage by the RE, turned to the radiomimetic antibiotics BLM and NCS. These clasto as defined by RE-induced mutation in the APRT gene (Ref. 19; Fig. genic compounds react in concerted fashion to mediate site-specific 2). Although we verified the presence of only one genomic DSB, it is free radical attack on deoxyribose moieties in both strands of DNA, likely that many more DSBs were produced throughout the genome resulting in DNA DSBs of defined structure, in addition to SSBs and by RE treatment, and the frequency of complex aberrations observed closely opposed abasic sites (36). BLM produces strand breaks with in first-division metaphases supports this notion. Genomic instability 5'-phosphate and 3'-phosphoglycolate termini, and abasic sites was not observed after numerous treatments using four different REs, formed in roughly equal proportions (36â€”39). NCS-induced DNA each with qualitatively different termini. Table 2 shows that regardless damage produces DNA DSBs with 3'-phosphate and 5'-aldehyde or of whether mutations were RE-induced, none of the 233 clones 5'-carboxylic acid termini as well as abasic sites (36, 40â€”42).A small analyzed was found to exhibit delayed chromosomal instability. In fraction of the NCS chromophore is also found cross-linked to the terestingly, related work has found REs unable to induce novel aber DNA (43). rations at early divisions after treatment (31). Furthermore, data Analysis of 62 clones, surviving BLM concentrations which re suggest that mutation frequency as measured at the APRT locus does not correlate with delayed genomic instability. To investigatethecapacityof REsto modulateanotherendpoint of NCSNCS Table6 Dclayedchromosomalinstability induced by genomic instability, i.e., delayed reproductive cell death, RE-induced concentration of clones of metaphase aberrant and non-RE-induced APRT mutant clones were analyzed for persis (@zgIml)No.metaphasesâ€•1.0141â€”20â€”33.09analyzedNo. subpopulationsâ€•% tent reductions in plating efficiency. Results shown in Table 3 reveal no significant difference in the plating efficiencies measured between RE-induced and non-RE-induced mutants. These results are in con 2 1â€”2 100 1 5 100 trast to those reported by Chang and Little (32), who found REs I 9 99 capable of eliciting a persistent reduction in plating efficiency. The 10010.07 I1â€”2 >250â€”2 discrepancies noted above are likely the result of differences between 2 3â€”4 2â€”9 our two experimental systems. I 7 18 Exposure to ionizing radiation results in damage to nuclear as well I 22 100 I1 >350â€”2 as nonnuclear targets, in part through the action of hydroxyl radicals. 100 a Numberofdifferentabnormalmetaphasesubpopulationsshowingdistincttypesof To mimic the indirectactionof radiationon targetmolecules,without chromosome aberrations. the formation of DNA DSBs, we investigated the effects of H2O2, b Percentage of metaphases scored showing chromosomal aberrations. 4054

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. INDUCtiON OF DELAYED GENOMIC INSTABILITY sulted in at least 1.5 logs kill (Fig. 4), shows that BLM was able to 8. Holmberg. K., Falt, S., Johansson, A., and Lambert. B. Clonal chromosome aberra induce delayed genomic instability. The observed frequency of insta lions and genomic instability in X-irradiated human T-lymphocyte cultures. Mutat. Res., 286: 321â€”330,1993. bility was proportional to drug levels; raising the concentration of 9. Marder, B. A., and Morgan, W. F. Delayed chromosomal instability induced by DNA BLM from 100 to 250 @tg/mlmore than doubled the percentage of damage. Mol. Cell. Biol., 13: 6667â€”6677,1993. 10. Martins, M. B., Sabatier, L., Ricoul, M., Pinton, A., and Dutrillaux, B. Specific unstable clones from 21 to 58% (Table 5). Many of these clones (total, chromosomeinstabilityinducedby heavyions: a step towardstransformationof severi) were markedly unstable, with five or more aberrant popula human fibroblasts? Mutat. Res., 285: 229â€”237, 1993. lions predominating the net population. The complexity of these I I. Kadhim, M. A., Lorimore, S. A., Hepburn, M. D.. Goodhead. D. T.. Buckle. V. J.. and Wright, E. G. a-Particle-induced chromosomal instability in human bone marrow rearrangements is illustrated in Fig. 5, which depicts a fraction of the cells. Lancet, 344: 987â€”988,1994. aberrations detected in an unstable clone classified to contain over I6 12. Sabatier, L., Lebeau, J., and Dutrillaux, B. Chromosomal instability and alterations of distinct rearranged populations after a l00-@.&g/m1treatment of BLM. telomeric repeats in irradiated human fibroblasts. mt. J. Radiat. Biol., 66: 61 1â€”613, I994. Similarly, analysis of 40 clones isolated after NCS treatment 13. Ward, J. F. DNA damage produced by ionizing radiation in mammalian cells: indicates that NCS also elicits delayed genomic instability (Table identities. mechanisms of formation, and reparability. Prog. Nucleic Acids Res. Mol. 6). However, only those clones isolated after I.5-log kill (>3.0 Biol.,35:95â€”125,1988. 14. Ward, J. F., Limoli, C. L., Calabro-Jones, P. M.. and Aguilera, J. An examination of @.tg/mlNCS; Fig. 6) exhibited genomic instability. As with BLM, the repair saturation hypothesis for describing shouldered survival curves. Radiat. the observed frequency of instability was proportional to drug Res., 127: 90â€”96, 1991. IS. Limoli. C. L., and Morgan. W. F. Genomic instability: Initiation and perpetuation. levels (Table 6). Of the eight unstable clones identified, six had Tenth International Congress on Radiation Research. Wurzburg: UniversitSts five or more aberrant populations representing the majority of all druckerei H. Sttlrtz AG, 1995. metaphases scored. Of these, three were remarkably unstable, 16. Adair, G. M., Carver, J. H.. and Wandres, D. L. Mutagenicity testing in mam malian cells. I. Derivation of a Chinese hamster ovary line heterozygous for the where all metaphases examined were aberrant with well over 20 adenine phosphoribosyltransferase and thymidine kinase loci. Mutat. Res., 72: distinct subpopulations of rearrangements identified within each 187â€”205,1980. clonal population. 17. Thompson, L. H., Fong. S., and Brookman, K. Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster Our findings that delayed genomic instability can be induced by cells. Mutat. Res., 74: 21â€”36,1980. exposure to BLM and NCS demonstrate, for the first time, the per 18. Bradley. W. E., and Letovanec, D. High frequency nonrandom mutational event at the adenine phosphoribosyltransferase (aprt) locus of sib-selected CHO variants het sistent destabilization of chromosomes following chemical treatment. erozygous for aprt. Somatic Cell Genet., 8: 51â€”66,1982. DNA damage in the form of complex DSBs may constitute at least 19. Phillips. J. W.. and Morgan, W. F. Illegitimate recombination induced by DNA one of the signals that initiate the onset of genomic instability. As with double-strand breaks in a mammalian chromosome. Mol. Cell. Biol.. 14: 5794 â€”5803, 1994. ionizing radiation, DSB damage resulting in both 3' (BLM) and 5' 20. Miller. S. A., Dykes. D. D., and Polesky. H. F. A simple salting out procedure for (NCS) chemical modification of DNA termini require some degree of extracting DNA from human nucleated cells. Nucleic Acids Res., 16: 1215. enzymatic processing before gap filling and strand resealing. Such 1988. 2 I. Tucker, J. D., Morgan. W. F.. Awa, A. A.. Bauchinger. M.. Blakely. D., Cornforth. lesions may challenge cellular repair, as opposed to the readily rep M. N.,Littlefield,L.G., Natarajan,A.T.. andShasserre.C.A proposedsystemfor arable termini formed by restriction enzymes and single-strand nicks scoring structural aberrations detected by chromosome painting. Cytogenet. Cell induced by H202. Genet., 68: 211â€”221. I995. 22. Adair, G. M., Stallings, R. L., Nairn. R. S.. and Siciliano, M. J. High frequency Our results suggest that any agent capable of producing complex structural gene deletion as the basis for functional hemizygosity of the adenine types of DNA DSBs will induce genomic instability. The frequency phosphoribosyltransferase locus in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA, 80: 5961â€”5964. 1983. with which genomic instability is observed, particularly after ionizing 23. Adair, G. M., Stallings, R. L., and Siciliano, M. J. Chromosomal rearrangements and radiation or treatment with radiomimetic antibiotics, suggests multiply gene expression in CHO cells: mapping of alleles for eight enzyme loci on CHO redundant pathways for induced instability. Our data using RE suggest chromosomes 13, Z4, Z5, and Z27. Somatic Cell Mol. Genet., 10: 283â€”295,1984. 24. Dewyse, P.. and Bradley, W. E. Loss of alleles in aprt mutants of CHO cells that DNA double-strand breakage per se does not necessarily lead to demonstrated by BcII restriction-fragment-length variation. Somatic Cell Mol. Gen chromosomal instability. Furthermore, DNA lesions induced by et., 16: 225â€”230,1990. H2O2, which can be lethal to a cell, do not appear to lead to instability. 25. Adair, 0. M., Siciliano, M. J., Brotherman, K. A.. and Naim, R. S. Preferential loss or inactivation of chromosome Z4 APRT allele in CHO cells. Somatic Cell Mol. Instead, our data indicate that the complexity or quality of DNA DSBs Genet., /5: 271â€”277,1989. may be important in initiating chromosomal instability. Other down 26. Adair, G. M., Naim, R. S., Brotherman, K. A., and Siciliano. M. J. Spontaneous CHO APRT heterozygotes reflect high-frequency. allele-specific deletion of the chromo stream cellular processes that occur in response to DNA damage may some Z4 APRT gene. Somatic Cell Mol. Genet.. 15: 535â€”544,1989. modulate further the extent, frequency, and heterogeneity of genomic 27. Bender, M. A., Awa, A. A.. Brooks, A. L., Evans, H. J., Groer. P. G., Littlefield, instability and can include damage-induced induction of genes pro L. G., Pereira, C., Preston, R. J., and Wachholz, B. W. Current status of cytogenetic procedures to detect and quantify previous exposures to radiation. Mutat. 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Charles L. Limoli, Mark I. Kaplan, John W. Phillips, et al.

Cancer Res 1997;57:4048-4056.

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