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[C AN CER RE SEA RC H 33, 961-965, M ay 1973]B leomycin-induced Chromosomal Aberrations in CulturedMammalian C ells1Kam la D. Paika and A wtar K rishan2Childr en 's C an ce r R esea rc h F ou nd atio n [K . D . P ., A . K ./, a nd D ep artm en t o f P ath olo gy [A . K ./, H arv ar dM e dica l Sc ho ol, B os to n, M as sa ch usetts02115

SUMMARYMouse fibroblasts (L-929) and human cells (HeLa andCCRF-CEM lym phoblasts) exposed to bleom ycin in vitro for20 hr showed a marked inhibition in the number of cellse nte rin g m ito si s. B le omycin -in duced chr omosomal a be rr ati on swere seen in cells from the three cultured lines and werem ostly in the form of chrom atid gaps, breaks, and fragm ents,and m ultiradial translocations. H um an lym phoblasts (C CR F-CEM ) were m ore resistant to the bleom ycin-induced m itotic

inhibition and chromosomal aberrations than the L-929 orHeLa cells. A small number of chromatid fragments andtranslocations w ere also seen in cells exposed to bleom ycin inthe G 2 or post-D NA -synthetic part of the cell cycle.INTRODUCTIONBleomycin, a new antitumor antibiotic isolated fromm utant strains of Streptom yces verticillus by Um ezawa et al.(18), is a com plex glycopeptide (2, 18). B leom ycin-inducedgrowth inhibition of Escherichia coli and HeL a cells in vitroand of Ehrlich's carcinom a and Sarcom a 180 cells in vivo has

been reported (17). In E . coli and HeLa cells, bleomycininhibits uptake of DNA precursors, but it has no inhibitoryeffect on the uptake of precursors for RNA and proteinsynthesis (17). Binding of bleom ycin to single- and double-stranded DNA has been suggested as a possible mode for thecy to to xic a ctiv ity o f th is c om po und (1 3,1 5).C hrom osom al aberrations induced by bleom ycin have beenreported both in vivo (3) and in vitro (11). In short-termhuman leukocyte cultures exposed to bleomycin, a highincidence of chromosomal abnormalities in the form ofchrom atid gaps, breaks, and fragm ents, and translocationswere reported by Ohama and Kadotani (11). Similar chromosomal aberrations, which in some cases persisted up to 1month posttreatment, were reported in the bone marrow ofp atien ts treated w ith b leomy cin (3 ).In cultured Chinese hamster ovary cells, bleomycin killscells in m itosis; a decreasing order of sensitivity is shown bycells in G 2, S , and G respectively (1). T obey (16) reportedthat in the C hinese ham ster cell line bleom ycin had little or no'T his study w as supported in part by R esearch G rants C -6516 fromthe N ational Cancer Institute and FR-05526 from the Division ofRe se ar ch Fac il it ie s a nd Re sour ce s, N IH.2To w hom reprint requests should be sent, at C hildren's C ancer

R es ea rc h Fou nd at io n, 3 5 B in ne y S t., B os to n, Ma ss . 0 21 15 .R ec ei ve d Aug us t 1 6, 1 97 2; a cc ep te d J an ua ry 2 9, 1 97 3.

effect on DNA synthesis and that the treated cells accumulated irreversibly in the G2 part of the cell cycle. Intransplantable VX-2 rabbit carcinoma cells exposed tobleomycin, the original population of stem-line cells isreplaced by cells with 2 to 4 tim es m ore DN A content (10).Bleom ycin has been used in the chemotherapy of a varietyof hum an tum ors, like squam ous cell carcinom as of the skin,head, neck and lungs, as well as against melanomas,lym phom as, and H odgkin's disease (7, 8, 12, 14).In view of the possible clinical im portance of bleom ycin in

the chem otherapy of a variety of hum an neoplasia, this studywas undertaken to evaluate the effect of this new drug onchromosomes of cells from cultured mammalian cell lines,including a hum an lym phoblastic cell line initiated from thep erip heral b lo od o f a leu kem ic p atien t.M ATERIALS AND METHODSEarle's L-929 (m ouse fibroblasts) and HeLa cells (hum an

cervical carcinoma) were grown as monolayer cultures andnourished with Eagle's minimum essential medium supplem ented with 10% fetal calf serum . Hum an leukem ic lym phoblasts, CCRF-CEM (6), derived from the peripheral bloodbuffy coat of a child w ith acute lym phoblastic leukem ia, w eregrown in suspension cultures in Eagle's m inim um essentialm ed ium su pplem en ted w ith 10% fetal calf serum . A ntibiotics,penicillin (100 i.u./m l), and streptom ycin (100 M g/m l) w ereadded to the culture media. Culture medium was changed 24hr prior to the harvesting of cells for the cytogenetic studies.Bleom ycin (Lot No. 71L 444; Bristol Laboratories, Syracuse, N .Y.) was dissolved in tissue culture medium (0.25 to100 (g /ml ),and cultures w ere treated for 4 or 20 hr. C olcem id(0.05 M g/m l) or vinblastine sulfate (0.01 M g/m l) w as added tocultures 4 hr before termination of incubation with bleomycin. For assessment of the effect of bleomycin on L- andHeLa cells in the G2 (post-DNA-synthetic) part of the cellcycle, m itotic plates from cultures exposed concom itantly tobleom ycin and C olcem id for 4 hr w ere used. Radioautographsm ad e from th ese C olcem id -arrested m ito tic cells from cu ltureslabeled with thymidine-3H showed that none of the mitoticcells were synthesizing DNA in the 4-hr period prior toharvesting and were thus in the G2 part of the cell cycle.M onolayer cultures w ere scraped w ith a rubber policem an,a nd s usp en sio n c ultu re s w ere c olle cte d b y lig ht c en trifu ga tio n.Cell pellets w ere w ashed in H anks' balanced salt solution andincubated in hypotonie 1% sodium citrate for 30 min at 37.C ell p ellets w ere retriev ed b y ce ntrifu gatio n an d fix ed in acetic

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K am la D . P aika a nd Aw ta r K rish anacid:alcohol (1:3) for 30 min. A ir-dried preparations weres ta in ed in C arb ol:fu ch sin (4 ). O nly w ell-sp re ad a nd a pp are ntlyintact m etaphase plates w ere analyzed from each sam ple forpossible drug-induced chrom osom al changes. F or the estim ation of m itotic index, an average of 1000 cells from air-driedand stained preparations or acetocarmine squashes werecounted.RESULTSEarle 's L-929 Cells. M itotic index data in Table 1, Colum n

1, show that exposure of L-cells to concentrations ofbleomycin above 1 f/g/ml for 20 hr causes a significantinhibitory effect on the entry of cells into m itosis. In culturesexposed to bleomycin, 25 M g/ml, for 20 hr (including 4 hr oftreatment with Colcemid for arrest of mitotic cells), themitotic index was only 6%. A significant increase in thenumber of chromosomal aberrations was seen with theincrease in the concentration of bleom ycin (Table 1, Colum n1 1), reach in g a m ax im um o f 1 7 ab erration s/1 00 ch romo somesexam ined in cells exposed to bleom ycin, 10 /ng/m l, for 20 hr.In cultures exposed to bleomycin, 25 tg/ml(which had alow er m itotic index of 6% ), there w ere few er aberrations thanin cells exposed to 10-//g/m l doses of the drug.Bleom ycin-induced chrom osom al aberrations seen w erem ostly in the form of translocation m ultiradials, chrom atid

breaks, and chromosomal fragments; these are also listed inTable 1, Columns 4 to 9. Arrows in Fig. 1 point to a numberof chromosomal translocations and chromatid breaks inch romosomes from a cell ex posed to b leomy cin , 1 0 /g /m l,fo r20 hr.To analyze the effect of bleomycin on cells in thepost-D NA -synthetic part of the cell cycle, w e studied m itoticplates from cultures exposed concom itantly to bleom ycin andColcem id for 4 hr; as seen in Table 1,20 plates analyzed fromthese cultures had a total of 21 chromosomal aberrations,which were m ostly in the form of chrom atid breaks, gaps, andfragmen ts. N o translo cation s (freq ue ntly se en in ce lls ex po sedto bleom ycin for 20 hr) w ere seen in these cultures.He La Cells. Data in Table 1 show that HeLa cells wereslightly more sensitive to the mitotic inhibitory actions ofb leomycin th an L -cells. W hereas in u ntre ated co ntro l c ulturesm itotic index was 29% , in cultures treated w ith bleom ycin, 1 /g /m l,it w as 1 4% ; w ith in cre as e in b le omyc in c on ce ntra tio n,the mitotic index dropped to as low as 2.5% in cells exposedto 100 /ug/ml for 20 hr. An initial increase in the number ofchromosomal aberrations seen with the increase in theconcentration of bleom ycin w as follow ed by a decrease in thenum ber of such aberrations in cultures exposed to higher dosesof the drug and with a lower m itotic index.B leomy cin -ind uced ch romo somal ab erratio ns seen in H eL acells (Fig. 2) were similar to those seen in L-cells (Table 1).B esides these aberrations, a num ber of H eL a plates (approxi-

T able 1Chromos omal a be rr at io ns i n L -9 29 , H eL a, a nd CCRF -CEM c ell s e xp os ed t o v ar io us c on ce ntr ati on s/t im e o f b le omyc inChromosoma l aber ra tions

L-929cellsHeLacellsCCRF-CEMcellsBleomycinconcentration/timeUntreated10.0

Mg/4i0.25fig/20i0.50Mg/20i0.75Mg/20i1.0ng/20r5.0Mg/20i10.0Mg/20r25.0Mg/20rUntreated25.0Mg/4hr50.0Mg/4r1.0Mg/20r10.0Mg/20i25.0Mg/20i50.0Mg/20i100.0Mg/20rUntreated1.0

Mg/20r10.0Mg/20r25.0Mg/20i50.0Mg/20r100.0Mg/20hrMitotic

index"21212322201210629148.97.88.12.526212017184.5No.ofplatesan alyzed64020373738251554202618163745444026203030768025Total

no. ofchromosomes260022852108206720081453692262011991746119711552322247523232349977908150815083matidgaps06503116201212111653970706816218Chromatidbreaks09424668143114500101344100145157340912817621Chromatidfragments85162921146151193501911181572900816296Deleions010004202001072091002333Ringchromosomes00015270781000000001011000Trans-ocations1001724311113637123133122418

wiiiiextensivechromosomefragmentation00000204002022855

uiaberrat ions/ 100chromosomes0.311.

1R ep re se nt s p er ce nt ag e o f m eta ph as e c el ls a fte r e xp os ur e o f c ul tu re s to Col cemi d o r v in bl as ti ne f or 4 h r.b M i to tic p la te s w ith e xte ns iv e f ra gm en ta ti on o f c hr omos omes a re n ot i nc lu de d.

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B leomyc in -i nduc ed Chromosoma l Abe rr ati on sm ately 13% ) from cultures exposed to m ore than bleom ycin,10 (g/m l, showed extensive fragm entation of the chrom osoma l compl emen t.In m itotic plates from H eL a cultures exposed to bleom ycinand Colcem id for 4 hr and show n by radioautographic studiesto belong to the post-DNA-synthetic (G2) part of the cellcycle population, a num ber of chrom osom al aberrations in theform of chromosomal fragments and chromatid breaks wereseen (F ig. 3). A sm all num ber of translocations w ere also seenin these cells as tabulated in T able 1, C olum n 9.CCRF-CEM Cells. In contrast to L-929 and HeLa cells,human leukemic lymphoblasts were more resistant to thebleom ycin-induced chrom osom al dam age and m itotic inhibition (Table 1). A significant drop in mitotic index was seenonly in cultures exposed to very high (100 /Jg/m l) concentrations of bleom ycin for 20 hr.A lth ou gh b le omyc in -in du ce d c hromo soma l a be rra tio ns se enin CCRF-CEM cells were sim ilar to those in L- and HeLa cells,they w ere seen w ith lesser frequency, as show n in T able 1. Fig.4 shows a mitotic plate from a CCRF-CEM cell exposed tobleomycin, 50 Mg/ml, for 20 hr. It shows a number ofc hromo soma l d ele tio ns, c hroma tid b re ak s, a nd a tra ns lo ca tio n.DISCUSSIONIn ba cteria (E . co li) an d m ammalia n cells (H eL a), b leomyc inh as b een repo rted to cau se sin gle- and d oub le-stran de d scissio nof DNA but not that of rRNA or tRNA (13). Similarly,Terasima et al. (15) have shown that bleomycin inducessingle-stranded breaks in D NA of cultured m ammalian cells at1 0 Mg /m l a nd d ou ble -stra nd ed b re ak s a t h ig he r c on ce ntra tio nsof 20 M g/m l. In short-term hum an leukocyte cultures, O ham aan d K ad otan i (1 1) h av e rep orte d th e o ccu rren ce of b leomy cin -in du ce d c hromo soma l a be rra tio ns, e .g ., b re ak s, tra ns lo ca tio ns,and a very high incidence of acentric fragments. In bonemarrow of bleomycin-treated patients, Bornstein et al. (3)h av e sh ow n th e p resen ce o f sim ilar ch rom osom al aberration s,w hich p ersiste d fo r u p to 1 m onth p osttreatm en t.Present studies confirm that bleom ycin induces chrom osomal aberrations in cultured m ouse fibroblasts and hum ancells o f n eo plastic o rigin, e.g ., H eL a cells an d CCR F-C EM cells.The damage seen is mostly in the form of chromosomalfragments, ch romatid b reak s, an d tra nslo catio ns. C CR F-C EMlym phoblasts are m ore resistant to the bleom ycin-inducedchrom osom al dam age than the L -929 or H eL a cells.Some of the recent reports have described the cellcycle-dependent effect of bleomycin (1, 5, 10, 15, 16); forexam ple, T erasim a et al. (15) have suggested that the effect ofbleomycin on cell cycle traverse is sim ilar to that of X-ray.

T hat is, cells are m ost sensitive in late G ! (presynthetic) stageand less sensitive in the S and early d part of the cell cycle.N agatsu et al. (10) have show n that bleom ycin prevents visiblem itosis but does not inhibit D NA synthesis, thus resulting incell populations with 2 to 4 times the DNA content ofstem -line cells. Barranco and Hum phrey (1) have shown thatbleom ycin is m ost effective in killing C hinese ham ster ovarycells in mitosis, G2 S, and Gin a decreasing order ofeffectiven ess. T heir c ell cy cle traverse ex perim en ts sh ow th atcell progression is affected most severely by bleomycin at

mitosis and at G2, while the cells in G] and S progresssmoothly; their biphase dose-response curve suggests asensitive and a less sensitive population. Tobey (16), in hisstudies on the effect of bleom cyin on cell cycle traverse, hasreported very little effect of bleomycin on DNA synthesis;how ever, bleom ycin w as extrem ely effective in stopping cellsir re ve rs ib ly in G2 .In the present studies on bleom ycin-induced chrom osom alaberrations, we exposed each of the 3 cell lines to variousconcentrations of bleomycin for 20 hr. Thus, all thech rom osom al p rep aratio ns w ere from cells that h ad c om pleteda major part of the cell cycle in the presence of the drug. Theh ig h in cid en ce o f tran slo catio n m ultirad ials in L -9 29 cells an dtranslocations in HeLa cells suggests that most of thebleom ycin-induced chrom osom al dam age is in the D NA -syn-thetic or S phase of the cell cycle. However, in L-cells andHeLa cells exposed to bleomycin for only 4 hr (essentiallyG2), there is evidence of some chromatid breaks, chromosom al fragments, and a few translo cation s, th us su gg estin g th atpart of the damage may also be caused in the G2 part of thecell cycle .A d ifferen tial sen sitiv ity of v ariou s cell typ es to b leomyc inhas been reported by other workers (10, 15). Several reportsfrom clinical studies have shown that bleom ycin concentrations are higher in certain parts of the body, such as the skinand lungs. In a recent study, w e have reported variations in theex ten t o f bleom ycin-in du ced ultrastru ctu ral ch an ge s d ep ending on the cell type (9), and thse observations are furtherconfirmed by the present study. Bleomycin, 10 Mg/ml, isstrongly inhibitory (50% ) against the entry of H eL a cells intom itosis, w hereas in L -929 cells and C CR F-CEM cells a m arkedreduction in the mitotic cell number is seen only atc on ce ntra tio ns o f a bo ve 1 0 a nd 5 0 ig /m l, esp ec tiv ely .Bleomycin inhibits mitosis and induces chromosomalaberrations, m ostly in the S phase and to a lesser extent in theG 2 p hase o f th e cell cy cle, an d there is a d ifferential sen sitiv ityof the various cell types to the drug.ACKNOWLEDGMENTSWe are thankful to D r. H erbert L azarus and D r. G eorge E . F oley forcultures of C CR F-CEM cells. T he technical assistance of M s. SallyW hitlock, M s. L inda W arren, and M s. M arjorie K asac is gratefullyacknowledged.

REFERENCES1. Barranco, S. C ., and H um phrey, R . M . The Effects of Bleom ycino n Sur viv al a nd C ell P ro gr es sio n i n Chi ne se H am st er C el ls i n V itr o.Canc er Re s. , 3 1: 1 218- 1223 ,1 971.2. B leom ycin (B leom ycin H ydrochloride for Injection). T okyo,Ja pan : N ip pon K ay aku C o. L td.3. B ornstein, R . S ., H ungerford, D . A ., H aller, G ., E ngstrom , P . F .,and Y arbro, J. ,'.C ytog en etic E ffec ts o f B le om ycin T herap y inM an. C an ce r R es., 3 1: 2 004 -2 00 7, 19 71 .4. C arr, D . H ., and W alker, J. E . C arbol F uchsin as a S tain for H um anC hromo somes. S tain T ech no l., 3 6: 23 3-2 36, 1 96 1.5. D rewinko, B., Novak, J., and Barranco, S. C . The Response ofH um an Lym phom a Cells in V itro to B leom ycin and l,3-B is(2-c hl or oe th yl) -l -n it ro so ur ea . C an ce r R es ., 3 2: 1 20 6- 12 08 , 1 97 2.

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K am la D . P aika a nd Aw tar K rish an6. F oley, G . E ., L azarus, H ., F arber, S ., U zm an, B . G ., B oem e, B . A .,a nd McC arth y, R . E . C on tin uo us C ultu re o f H um an L ym ph ob las tsfrom P eripheral B lood of a C hild w ith A cute L eukem ia. C ancer,18: 522-529, 1965.7. Harada, T ., Nishikawa, Y., Suzuki, T., Ito, K ., and Baba, S.B leom ycin Treatm ent for Cancer of the T hyroid. A m. J. Surg.,722:53-57, 1971.8 . Ich ik aw a, T . B le om ycin , a N ew A ntitu mo r A ntib iotic-as a S pec ificagainst Squam ous C ell C arcinom a. J. Japan. M ed. A ssoc., 61:487-497, 1969.9. K rishan, A . Bleom ycm -induced F ine Structural A lterations inC ultured M ouse Fibroblasts and H um an L ym phocytes of N eo-p la st ic O rig in . C an ce r R es ., 3 3: 7 77 -7 85 , 1 97 3.1 0. N ag atsu , M ., O ka ga ki, T ., R ic hart, R . M ., an d L am bert, A . E ffe ctso f B leomy cin o n N uclea r DNA in T ran sp lan ta ble VX-2 C arcin om aof R abbit. C ancer R es., 31: 992-996, 1971.11. O ham a, K ., and K adotani, T. C ytologie E ffects of B leom ycin onCultured Human Leukocytes. Japan. J. Human. Genet., 14:2 93-2 97 , 1 970 .

12. S hastri, S ., S layton, R . E ., W oher, J., P erlia, C . P ., and T aylor, S .G . C lin ic al S tu dy w ith B ie om ycin . C an ce r, 2 8: 1 14 2-1 14 6, 1 971 .13. S uzuki, H ., N agai, K ., A kutsu, E ., Y am aki, H ., and T anaka, N . O nthe M echanism of A ction of B leom ycin: S trand S cission of D NAC aus ed by B leomyc in a nd Its B in din g to DNA in V itro . J. A ntib io t.T okyo Ser. A , 2 3: 473-480, 1970.14. Takeda, K ., Sagaw a, Y ., and A rakaw a, T. T herapeutic Effect ofB le om ycin fo r S kin T um ors . G an n, 61 : 2 07 -2 18 , 1 97 0.15. Terasima, T., Yasukawa, M ., and Umczawa, H. Breaks andRejoining of DNA in Cultured M am malian Cells Treated withB le om ycin . G an n, 6 1: 5 13-5 16 , 1 970 .16. T obey, R . A . A S im ple, R apid T echnique for D eterm ination of theE ffects of C hem otherapeutic A gents on M ammalian C ell-C ycleT rav erse . C an ce r R es., 32 : 3 09 -31 6, 19 72 .17. Umezawa, H., Ishizuka, M ., and Takeuchi, T . Studies onB leomy cin . C an cer, 2 0: 8 91 -8 95 , 1 96 7.18. Umezawa, H., Maeda, K ., Takeuchi, T ., and Okam i, Y . NewA ntibiotics: B leom ycin A and B . J. A ntibiot. Tokyo Ser. A , 79:200-209, 1966.

Fig. 1. Part of a L-929 cell m etaphase plate from a culture exposed to bleom ycin, 10 M g/m l, f or 20 hr. A rrow s, m ultiple translocation andb re ak s. X 2 40 0.Fig. 2. M etaphase plate from a HeLa cell culture exposed to bleom ycin, 50 fig/m l, for 20 hr. Arrows, drug-induced gaps, breaks, andc hr omos omal f ra gm en ts . X 1 44 0.F ig . 3 . M etap has e p la te fro m a H eL a ce ll e xp ose d to b le om ycin , 5 0 n g/m i, for 4 h r an d sh ow ing c hromatid b rea ks a nd tran sloc ation . X 1 520 .F ig. 4. M etaphase plate from a C CR F-C EM culture treated w ith bleom ycin, 50 jug/m l, for 20 hr. A rrow s, chrom atid breaks, deletions, and at ra ns lo ca ti on . X 1560.

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B leomyc in -i nduc ed Chromosomal Abe rr at ions

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