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Rare chromosomal aberrations induced by vincristine
Rare C3n'omosomal Aberrations Induced by Vincristine Partial Endoreduplication and Pseudoendoreduplication, Segmentally Endoreduplicated Chromosomes, and Segmental Premature Chromosome Condensation Hideki Takanari, Koji Katsuta, Toshimichi Yoshida, Ryuichi Yatani, and Kosaku Izutsu
ABSTRACT: Vincristine [VCR) is capable of inducing a cell containing both conventional chromosomes (monochromosomes) and diplochromasomes. A total of 124 such metaphases were examined by 5-bromodeoxyuridine (BrdU) incorporation and fluorescence plus Giemsa (FPG) technique to analyze (:ell cycle kinetics. The majority of cells (119 metaphases) showed differential BrdU incorporation between the two kinds of chromosomes, indicating that partial endareduplication occurred in these cells. In addition, existence of partially endoreduplicated cells with premature chromosome condensation (PCC) in either mona- or diplochromosomes suggests that the timing of monochromosome-replication was very variable in individual cells. On the other hand, the remaining five metaphases showed that both mona- and diplochromosomes incorporated BrdU similarly, indicating that diplochromosomes are formed by pseudoendoreduplication. Two kinds of chromosomal aberrations probably caused by delay of I)NA synthesis on chromosome segments, segmental endoreduplication, and segmental PCC were also reported. Segmental endoreduplication was defined as endoreduplication that occurred on some segments (ff chromosomes. Out of 119 partially endoreduplicated cells, 3 contained a chromosome consisting of both m(mo- and diplochromosomal segments, indicating that the former segments missed one raund of DNA synthesis. Segmental PCC was defined as PCC restricted to only some segments of chromosomes. Two types of segmental PCC. segmental S-PCC and G2-PCC, were observed in VCR-induced ordinary polyploidy. Althaugh both segmental endoreduplication and segmental PCC occurred with very low frequency, these phenamena suggest that DNA synthesis was disturbed in same part of the nucleus. INTRODUCTION P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n (PCC) [1] is a w e l l - k n o w n p h e n o m e n o n that s h o w s a b n o r m a l t i m i n g of DNA s y n t h e s i s o c c a s i o n a l l y o c c u r r i n g in m u l t i n u c l e a t e c e l l s or c e l l s w i t h a m i c r o n u c l e u s . T h e m e c h a n i s m for p r o d u c i n g PCC is e x p l a i n e d From the' Department of Pathology, Mie University School of Medicine. Tsu. Mie. Japan.
Address reprint requests to: Hideki Takanari, M.D.. Department of Pathology, Mie University School of Medicine, 2-174 Edobashi, "/'su, Mie 514, Japan. Received July I"L 1989; accepted January 18, 1990.
155 © 1991 Elsevier Science Publishing Co.. Inc. v 655 Avenue at" the Americas. New York. NY 10010
Cancer (;enet Cytogenet 51:155 165 (19t,lll 0165-4608,'91/$03.50
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H. Takanari et al. as follows: either asynchronous starting of DNA synthesis [2, 3l or a difference in progression of DNA synthesis [4] between the sister nuclei of a multinucleate cell providing the condition in which one sister nucleus is still in the G1. S, or G2 phase when the other nucleus enters the mitotic phase. The former nucleus undergoes PCC when chromosome condensation factors are activated upon the entry of the other nuclei into mitosis [1]. Another c:ytogenetic: phenomenon showing the abnormal timing of DNA synthesis is partial endoreduplication [5, 6]. A metaphase consisting of a mixture of conventional chromosomes (monochromosomes) and diplochromosomes has been reportect in a patient with leukemia [5] and in human fibroblast culture [7]. Our previous report [6] has shown that partial endoreduplication occurred in VCR-induced polyploid cells. Cell cycle kinetic study of VCR-induced partially endoreduplicated ceils revealed that diplochromosomes incorporated BrdU twice while monochromosomes did so once. There is a possibility that partial pseudoendoreduplication may also produce metaphases containing two kinds of chromosomes. The term partial pseudoendoreduplication has been used to describe a mechanism for producing a triradial c:hromosome [8]. However, corresponding to partial endoreduplication described above, partial pseucloendorecluplication is more likely to be defined as diplochromosome formation in some c:hromosomes, which failed chromatid separation in the previous mitosis and underwent an additional DNA replication. By this definition, there is little evidence for partial pseudoendoreduplication. One of the purposes of this study was to examine whether or not partial pseudoendoreduplication produced a metaphase containing two kinds of chromosomes and also to show more characteristics of partially endoreduplicated ceils. Both PCC and partial endoreduplication involve whole chromosome(s), reflecting disturbance in regulation of DNA synthesis in sister nucleus. However, Zur Hausen [9] has reported decondensed long arms of #1 chromosome in an established cell line, and has suggested that the disturbed DNA replication pattern seems to lead to inc:omplete condensation. These abnormal chromosomes are different from decondensed chromosomes indu('ed by various (:llemical agents [10-17]. This report represents other evidence showing the (tisturbance of DNA synthesis on chromosome segments.
MATERIALS AND METHODS Cell Culture CHO-kl cells were grown in Ham's F12 medium (Flow Laboratories) supplemented with 20% fetal calf serum (M.A. Bioproducts). Cells were seeded in T-25 plastic flasks and used for experiments 24 hours after subculturing. Cells were treated with 5 p.M vincristine (VCR, Sigma) for 12 hours. After they were washed twice in serum free medium, cells were recultured in fresh medium containing 10/~g/ml of 5-bromodeoxyuridine (BrdU, Wako) in the dark. The control cultures were also recultured in BrdUcontaining medium. Chromosome preparations were made from separate flasks at 30, 36, 42, and 48 hours after reculturing. In one series, BrdU was added 12 hours before VCR treatment.
Chromosome Preparation and Staining Chromosome preparations were done by standard air-dry method. Slides were stained by the FPG method as described earlier [6].
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Autoradiographic Study VCR-treated cells were recultured in fresh medium after washing. At 24 hours postrelease from drug, tritiated thymidine (specific activity 2 Ci/mol, ICN Radiochemicals) was introduced into the cultures at a final concentration of 3 ~Ci/ml. Chromosome preparations were made at 18 or 24 hours thereafter. Cells were fixed in a 1 : 1 mixture of acetic acid and methanol and then in a 3 : 1 mixture. The preparations were stained with Giemsa (Merck). Suitable metaphase figures were photographed. After they were destained with methanol, the slides were covered with Sakura NR-M2 emulsion film (Konishiroku Photo Ind. Co. Ltd.) and allowed to expose for 2 weeks. After developing, the slides were restained with Giemsa. The cells previously photographed were rephotographed.
RESULTS AND DISCUSSION
Partial Endoreduplication A total of 278 and 1,021 cells containing diplochromosomes were examined from three sets of the control and VCR-treated cultures, respectively. The frequency of such cells harvested at four time periods was 0.1%-0.3% in control and 1.1%-1.3% in VCR-treated cultures. No metaphase with a mixture of mono and diplochromosomes was observed in the control cultures, whereas 124 cells (12%/ in VCR-treated ones had metaphase with two kinds of chromosomes. The 124 metaphases containing two kinds of chromosomes were classified into three types in the FPG staining pattern (Fig. 1). In type I, all chromatids were darkly stained with Giemsa (Fig. la), indicating that both mono- and diplochromosomes incorporated BrdU once. In type II, two chromatids of each monochromosome and the two inner chromatids of each diplochromosome were stained darkly, while the two outer chromatids of diplochromosomes were pale (Fig. lb), indicating that only diplochromosomes underwent two rounds of DNA replication. In type III, each chromosome of both mono- and diplochromosomes consisted of two different kinds of chromatids, one unifilarly substituted with BrdU and the other bifilarly substituted (Fig. lc), indicating that both mono- and diplochromosomes incorporated BrdU twice. Table 1 shows the distribution of FPG staining pattern of the abnormal metaphases at each harvest time. More than 95% of the metaphases with two kinds of chromosomes were of type II staining pattern. The following two additional experiments were carried out to confirm the partial endoreduplication cycle. First, in order to know the distribution of original strands, BrdU was introduced into the cultures 12 hours before VCR treatment. All metaphases containing two kinds of well-condensed chromosomes showed the type IV FPG staining pattern: namely, one chromatid out of two and four chromatids in each mono- and diplochromosome, respectively, was stained with Giemsa's stain (Fig. ld). Second, in order to trace the timing of monochromosome replication, ]3H]-thymidine was introduced into the cultures during the last 18 or 24 hours before the harvest because a pulse treatment with [3H]-thymidine for less than 12 hours before the harvest failed to label partially endoreduplicated cells. The late replicating regions [11, 18-22] of both mono- and diplochromosomes were labeled. These data from the additional experiments suggest that endoreduplication started on some chromatids of VCR-induced polyploids and the remaining chromatids replicated once during diplochromosome formation. In fact, analysis of 28 well-spread, partially endoreduplicated metaphases revealed that the mean number of total chromosomes and of diplochromosomes found in each metaphase were 37.9 (range 29-48) and 10 (range
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T h e D i s t r i b u t i o n of F P G - s t a i n i n g Patterns of 124 Cells C o n t a i n i n g both M o n o c h r o m o s o m e s and D i p l o c h r o m o s o m e s at Each Harvest T i m e Hrs after removal of drug
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Partially endoreduplicated metaphases containing chromosomes showing PC(. (a) Monochromosomes show S-PCC. (b) Diplochromosomes show G2-PCC.
2-17), respectiw,qy, indicating that partial endoreduplication had occurred in neartetrallloid cells. Of 119 partially endoreduplicated cells, 31 (:ells had some chromosomes, either mono- or diplochromosomes, with varying degrees of PCC as shown in Fig. 2. These figures suggest that either set of monochromosomes or diplochromosomes differs in the DNA synthesis pattern; monochromosome replication did not finish until the end of diplochromosome replication (Fig. 2a) or it finished during the second DNA synthetic period of endoreduplicated chromosomes (Fig. 2b). In the former case, as an entrance into the S phase is inducible and as the endoreduplication is considered to be twice as long as the arbitrary cell cycle, markedly delayed monochromosome replication was very surprising. However, it is clear that the initiation signal for DNA synthesis activated one sister nucleus of a multinucleate cell and this nucleus formed diplochromosomes, while the other sister nucleus did not respond well to the stimulation for a long time. This raises the question: What regulates such differences in responsiveness to the stimulation of DNA synthesis? Solving this problem may elucidate the process of partial endoreduplication in detail. Partial Pseudoendoreduplication It is well known that mitotic inhibitors induce aneuploidy probably by mitotic nondisjunction [21-24]. In this case, if the nondisjunction chromosome keeps a centromeric connection, it is considered that the unseparated chromosomes may form diplochromosomes after another round of I)NA synthesis. Indeed, Figures la and 1(: show tile possibility that such a l)henomenon has occurred. In Figure la. 25 chromosomes int:htding seven diph)chromosomes were observed, indit:ating that the cell originated from VCR-induced aneuploidy. Tile existancc of (Jells with the type Ill BrdU-incorporation pattern (:onfirmed indire(:tlv that unseparated (:hronmsonles were induced by the treatment of mitotic inlaibitor. As described above, at least, two mechanisms could produce cells containing both mono- and diplochromosomes in the cultures treated with VCR (Fig. 3). Aneuploidy is formed by VCR-induced nondisjunction (upper line in Fig. 3). A centromeric connection between two sister chromatids leads to nondisjunction of some chromosomes, but a set of chromosomes is enclosed in the same nucleus. If such a nucleus undergoes another DNA synthesis in the presence of BrdU, the type I cell will result.
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In this mechanism, both mono- and diplochromosomes replicate synchronously. The second mechanism is partial endoreduplication. If one sister nucleus in a VCRinduced multinucleate cell undergoes two rounds of DNA syntheses, whereas the other does so once, the type II cell may be obtained (lower line in Fig. 3).
Segmentally Endoreduplicated Chromosomes in Partially Endoreduplicated Cells Out of 119 partially endoreduplicated cells, 3 cells contained at least one abnormal chromosome (Fig. 4a,c,e). The abnormal chromosome seems to consist of mono- and diplochromosomal segments (Fig. 4b,d,f). Two possible explanations, artifact and segmental endoreduplication, could be considered for the interpretation of these abnormal chromosomes.These chromosomes are actually diplochromosomes, and the monochromosomal segments are formed by two chromatids overlapping during the chromosome preparation. In general, some diplochromosomes, particularly small chromosomes, occasionally show such an overlapping appearance because diplochromosomes are located in three-dimensional fashion within the cell [25]. The overlapped chromatids, indeed, stained darkly and widely as the abnormal segments shown in Fig. 4, comparect with the usual chromatids. However, the chromosome condensation of such overlapped chromatids was similar to that of other normal diplochromosomes or nonoverlapped chromatids. Figure 4a,e show that apparently monnchromosomal segments of the abnormal chromosome condensed more than diplochromosomal segments did. Moreover, the condensation of monochromosomal segments was almost identical to that of conventional chromosomes observed in the same metaphase plate, indicating that the replication and chromosome condensation were coordinated between monochromosomes and monochromosomal segments of the abnormal chromosome in each cell. Therefore, we assumed these chromosomes were formed by delay of DNA replication on some segments of diplochromosomes. Thus, both the process of forming these abnormal chromosomes and their existance in partially endoreduplicated cells are unlikely to be correctly termed "segmental endoreduplication." However, the chromosome itself is segmentally endoreduplicated.
161
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Segmental PCC Segmental PCC is defined as PCC restricted to some segments of a chromosome. Two types of segmental PCC, segmental S-PCC and G2-PCC, were observed at 48 hours postrelease after VCR treatment, although their frequencies were as low as a few cells on one slide (about 2,000 mitotic cells). Chromosomes with segmental S-PCC were easily identified from the remaining chromosomes that were completely organized,
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as shown in Figure 5a. Karyotypic analysis of the cell shown in Figure 5a revealed pulverization of about four fifths of the short arm of X chromosome with slight organization at terminal segments (Fig. 5b). The reason why the replicated terminal segments u n d e r c o n d e n s e and the reason why the lower end of unreplicated segment attaches to chromatid with bifilarly substituted with BrdU are unknown. Figure 5 shows another example of segmental S-PCC of X chromosome. These are very similar to chromosomes presented by Zur Hausen [9] in the sense of u n d e r c o n d e n s a t i o n of some segments in one chromosome. However, in this case, u n d e r c o n d e n s a t i o n was restricted to both long arms of chromosome 1 while in our case, u n d e r c o n d e n s a t i o n occurred on one of the X chromosomes. Two major mechanisms which have been proposed for PCC in the multinucleate cells, a disturbance in the initiation of DNA synthesis (2, 3) or a disturbance in the progression of DNA synthesis (4), may be available for segmental PCC. In addition, it is also possible that these localized PCCs could be provoked by some abnormalities at the replication site, which consists of DNA strands and protein structures. Figure 6 shows an example of segmental G2-PCC. In Figure 6a, a large submetacentric c h r o m o s o m e undercondenses at terminal segments. Karyotypic analysis revealed that the abnormal chromosome was chromosome 1 (Fig. 6b). Figure 6c shows a large ring c h r o m o s o m e with u n d e r c o n d e n s e d segments of about one third. The u n d e r c o n d e n s a t i o n of some segments may occur by two possible mechanisms; inhibition of chromosome condensation or reflection of delay of DNA replication. The first mechanism, inhibition of chromosome condensation, is well documented: several chemical agents that are incorporated into the chromosome [10-15] or that bind to DNA strand [16, 17] have been found to inhibit chromosome condensation. In these cases, the segmental extension occurs in the specific regions. Late-replicating regions are u n d e r c o n d e n s e d in the first-round metaphase of diploid cells after short
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Figure 6 Chromosomes with segmental G2-PCC. {a) Hypotetraploid cell contained a chromosome with undercondensed segments. (b) A partial karyotype of the cell shown in Fig. 6a revealing that distal segments of short arm of chromosome 1 undercondensed. (c) A part of metaphase containing large-ring chromosome with undercondensed segments. treatment (during late S phase) with BrdU [10-12] or with 5-azacytidine [13-15[. Centromeric [16, 17] or G-C rich [14] heterochromatins are involved by Hoechst 33258 or 5-azacytidine, respectively. The second mechanism for producing undercondensation of some segments of chromosome is segmental G2-PCC, which reflects the disturbance of DNA synthesis. Although the effect of long treatment with BrdU was not c o m p l e t e l y negligible, we assume these chromosomes were formed by the second m e c h a n i s m because the abnormal chromosomes shown in Figure 6 contain both early and late replicating regions and segments are not restricted to any specific regions. Comments
The term partial pseudoendoreduplicotion has been used to explain the mechanism for formation of triradial chromosomes [8], i.e., after chromatid breakage, some segments attach to the sister chromatid, then replicate and form a triradial chromosome. However, as the aberration occurs in segments of chromosomes, and Drets et al. [26] used the term selective segmental endoreduplication for their symmetrical triradials, segmental p s e u d o e n d o r e d u p l i c a t i o n may be a more suitable term for a triradial chromosome than a partial one. At present, the disturbance in regulation of DNA synthesis has been mainly investigated in multinucleate cells or cells with micronucleus, and it is generalized that PCC is a result of mitoti(: asynchr(my in these (:ells. However, the rare abnormalities restricted to a part of (:hromosome, such as segmental e n d o r e d u p l i c a t i o n and segmental PCC, suggest that a similar disturbance of DNA synthesis o(:(:urs within the nucleus. This further indicates that a disturbance at each step in regulation of DNA synthesis results in many different types of (:hromosomal aberration. Although the abnormal cells described here were rarely observed, they indicate that some minor abnormality occurred during either mitosis or DNA replication after VCR treatment. Also it is likely that such minor abnormalities are more easily detected in late-culture period than in early ones. The biologic effect of these delayed changes of the drug, primary or secondary, is unknown. However, these minor changes may
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p r o d u c e a n e u p l o i d cells. Based on this a s s u m p t i o n and the fact that various p h y s i c a l and c h e m i c a l c a r c i n o g e n s i n d u c e p o l y p l o i d y and m u l t i n u c l e a t e cells, it is suggested that these a b n o r m a l i t i e s may be i n v o l v e d to establish c h r o m o s o m e aberrations of c a n c e r c e l l s - - i n particular, the c h a n g e of the g e n o m e number. Further studies are n e e d e d to e l u c i d a t e the possibility.
Authors wish to thank Dr. T. M. Kou, Department of Molecular Pathology, and Dr. J. C. Liang. Department of Genetics, M. D. Anderson Hospital and Tumor Institute, for critical reading of this manuscript.
REFERENCES 1. Johnson RT, Rao PN (1970): Mammalian cell fusion: Induction of premature chromosome condensation in interphase nuch;i. Nature 226:717-722. 2. Gosh S, Paweletz N, Gosh I (1978): Mitotic: asynchrony of multinuc:leate cells in tissue culture. Chromosoma 65:293-300 3. Harves JP, Lopez-Saez JF, Gimenez-Martin G (1982): Multinuc:leate plant cell. II. Requirements for DNA synthesis. Exp (;ell Res 139:341-350. 4. De la Torre C, Gimenez-Martin G (1977): Regulation of cycle progression in plant cells. Cell Biol Int Rep 1:211-223. 5. Houston EM, Levin WC, Ritaman SE (1964): Endoreduplication in untreated early leukemia. Lancet ii:496-497. 6. Takanari H, Nakakuki K, Izutsu K (1985): Cytogenetic demcmstration of out-of-phase DNA synthesis in endoreduplicated CHO cells: Evidence for partial endoreduplication. Cytogenet Cell Genet 38:93-98. 7. Lejeune J. Berger R, Rethore MO (1966): Sur l'endoreduplication selective de certains segments du genome. CR Acad Sci (D)(Paris) 263:1880-1882. 8. Stahl-Mauge C, Hager HI.), Schroeder TM (1978]: The problem of partial endoreduplication. Hum Genet 45:45-51. 9. Zur Hausen H (1967): Chromosome changes of similar nature in seven establishect cell lines derived from the peripheral blood of patients with leukemia. ] Nat Cancer Inst 38:683-696. 10. Zakharov AF. Egolina NA (1972): Differential spiralization along mammalian mitotic c:hromosomes. 1. BUdR-revealed differentiation in Chinese hamster chromosomes. Chromosoma 38:341-365. o 11. Stubblefield E (1974): The kinetics of DNA replication in chromosomes. In: The Cell Nucleus, Vol. II. H Busch, ed. Academic Press, New York, pp.149-162. 12. Zakharove AF, Baranovskaya LI, Ibraimove A1, Benjusch VA, Demintseva VS, Oblapenka NG (1974): Differential spiralization along mammalian mitotic chromosomes. II. 5-Bromodeoxyuridine and 5-bromodeoxycytidine-revealed differentiation in human chromosomes. Chromosoma 44:342-359. 13. Fucik V. Michaelis R, Rieger R (1970): On the induction of segment extension and chromatid structural changes in Vicia Faba chromosomes after treatment with 5-azacytidine and 5azadeoxycytidine. Murat Res 9:599-606. 14. Viegas-Pequignot E, Dutrillaux B (1981): Detection of G-C rich heterochromatin by 5-azacytidine in mammals. Hum Genet 57:134-137. 15. Hori T (1983): Induction of chromosome decondensation, sister chromatid exchanges and endoreduplication by 5-azacytidine, an inhibitor of DNA methylation. Mutat Res 121:47-52. 16. Matsukuma S, Utakoji T (1978): Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258. Exp Cell Res 113:453-455. 17. Marcus M, Sperling K (1979): Condensation-inhibition by 33258oHoechst of centromeric heterochromatin in prematurely condensed mouse chromosomes. Exp Cell Res 123:406-411. 18. Hsu T( ] (1964): Mammalian chromosomes in vitro XVIII. DNA replication sequence in the Chinese hamster. J Cell Biol 23:53-62. 19. Crossen PE, Pathak S, Arrighi FE (1975): A high resolution of the DNA replication patterns
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of Chinese hamster chromosomes using sister chromatid differential staining technique. Chromosoma 52:339-347. Wolff S, Bodycote J, Rodin B (1978): Chromosomal isolabelling caused by three rounds of synthesis in late replicating regions. Chromosoma 69:179-183. Cox DM, Puck TT (1969): Chromosomal nondisjunction: The action of Colcemid on Chinese hamster cells in vitro. Cytogenetics 8:158-169. Kato H, Yoshida TH (1970): Nondisjunction of chromosome in a synchronized cell population initiated by reversal of Colcemid inhibition. Exp Cell Res 60:459-464. Satya-Prakash KL, Hsu TC, Wheeler WJ (1984): Metaphase arrest, anaphase recovery and aneuploidy induction in cultured Chinese hamster cells following exposure to mitotic arrestants. Anticancer Res 4:351-356. Hsu TC, Satya-Prakash KL (1987): Aneuploidy induction by mitotic arrestants in animal cell systems: possible mechanisms. In: Aneuploidy, Etiology and Mechanisms, VL Dellarco. PE Voytek, A Hollaender, eds. Plenum Press, New York, pp. 279-287. Takanari H (1985): Studies on endoreduplication. V. A three-dimensional scheme for diploand quadruple chromosomes and a model for DNA replication. Cytogenet Cell Genet 39:188-193. Drets ME, Cardoso JM, Delfino AH, Carrau J (1970): Familial normal/partial trisomy 16 with selective endoreduplication in malformed proband. Cytogenetics 9:333-350.
ABSTRACT: Vincristine [VCR) is capable of inducing a cell containing both conventional chromosomes (monochromosomes) and diplochromasomes. A total of 124 such metaphases were examined by 5-bromodeoxyuridine (BrdU) incorporation and fluorescence plus Giemsa (FPG) technique to analyze (:ell cycle kinetics. The majority of cells (119 metaphases) showed differential BrdU incorporation between the two kinds of chromosomes, indicating that partial endareduplication occurred in these cells. In addition, existence of partially endoreduplicated cells with premature chromosome condensation (PCC) in either mona- or diplochromosomes suggests that the timing of monochromosome-replication was very variable in individual cells. On the other hand, the remaining five metaphases showed that both mona- and diplochromosomes incorporated BrdU similarly, indicating that diplochromosomes are formed by pseudoendoreduplication. Two kinds of chromosomal aberrations probably caused by delay of I)NA synthesis on chromosome segments, segmental endoreduplication, and segmental PCC were also reported. Segmental endoreduplication was defined as endoreduplication that occurred on some segments (ff chromosomes. Out of 119 partially endoreduplicated cells, 3 contained a chromosome consisting of both m(mo- and diplochromosomal segments, indicating that the former segments missed one raund of DNA synthesis. Segmental PCC was defined as PCC restricted to only some segments of chromosomes. Two types of segmental PCC. segmental S-PCC and G2-PCC, were observed in VCR-induced ordinary polyploidy. Althaugh both segmental endoreduplication and segmental PCC occurred with very low frequency, these phenamena suggest that DNA synthesis was disturbed in same part of the nucleus. INTRODUCTION P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n (PCC) [1] is a w e l l - k n o w n p h e n o m e n o n that s h o w s a b n o r m a l t i m i n g of DNA s y n t h e s i s o c c a s i o n a l l y o c c u r r i n g in m u l t i n u c l e a t e c e l l s or c e l l s w i t h a m i c r o n u c l e u s . T h e m e c h a n i s m for p r o d u c i n g PCC is e x p l a i n e d From the' Department of Pathology, Mie University School of Medicine. Tsu. Mie. Japan.
Address reprint requests to: Hideki Takanari, M.D.. Department of Pathology, Mie University School of Medicine, 2-174 Edobashi, "/'su, Mie 514, Japan. Received July I"L 1989; accepted January 18, 1990.
155 © 1991 Elsevier Science Publishing Co.. Inc. v 655 Avenue at" the Americas. New York. NY 10010
Cancer (;enet Cytogenet 51:155 165 (19t,lll 0165-4608,'91/$03.50
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H. Takanari et al. as follows: either asynchronous starting of DNA synthesis [2, 3l or a difference in progression of DNA synthesis [4] between the sister nuclei of a multinucleate cell providing the condition in which one sister nucleus is still in the G1. S, or G2 phase when the other nucleus enters the mitotic phase. The former nucleus undergoes PCC when chromosome condensation factors are activated upon the entry of the other nuclei into mitosis [1]. Another c:ytogenetic: phenomenon showing the abnormal timing of DNA synthesis is partial endoreduplication [5, 6]. A metaphase consisting of a mixture of conventional chromosomes (monochromosomes) and diplochromosomes has been reportect in a patient with leukemia [5] and in human fibroblast culture [7]. Our previous report [6] has shown that partial endoreduplication occurred in VCR-induced polyploid cells. Cell cycle kinetic study of VCR-induced partially endoreduplicated ceils revealed that diplochromosomes incorporated BrdU twice while monochromosomes did so once. There is a possibility that partial pseudoendoreduplication may also produce metaphases containing two kinds of chromosomes. The term partial pseudoendoreduplication has been used to describe a mechanism for producing a triradial c:hromosome [8]. However, corresponding to partial endoreduplication described above, partial pseucloendorecluplication is more likely to be defined as diplochromosome formation in some c:hromosomes, which failed chromatid separation in the previous mitosis and underwent an additional DNA replication. By this definition, there is little evidence for partial pseudoendoreduplication. One of the purposes of this study was to examine whether or not partial pseudoendoreduplication produced a metaphase containing two kinds of chromosomes and also to show more characteristics of partially endoreduplicated ceils. Both PCC and partial endoreduplication involve whole chromosome(s), reflecting disturbance in regulation of DNA synthesis in sister nucleus. However, Zur Hausen [9] has reported decondensed long arms of #1 chromosome in an established cell line, and has suggested that the disturbed DNA replication pattern seems to lead to inc:omplete condensation. These abnormal chromosomes are different from decondensed chromosomes indu('ed by various (:llemical agents [10-17]. This report represents other evidence showing the (tisturbance of DNA synthesis on chromosome segments.
MATERIALS AND METHODS Cell Culture CHO-kl cells were grown in Ham's F12 medium (Flow Laboratories) supplemented with 20% fetal calf serum (M.A. Bioproducts). Cells were seeded in T-25 plastic flasks and used for experiments 24 hours after subculturing. Cells were treated with 5 p.M vincristine (VCR, Sigma) for 12 hours. After they were washed twice in serum free medium, cells were recultured in fresh medium containing 10/~g/ml of 5-bromodeoxyuridine (BrdU, Wako) in the dark. The control cultures were also recultured in BrdUcontaining medium. Chromosome preparations were made from separate flasks at 30, 36, 42, and 48 hours after reculturing. In one series, BrdU was added 12 hours before VCR treatment.
Chromosome Preparation and Staining Chromosome preparations were done by standard air-dry method. Slides were stained by the FPG method as described earlier [6].
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Autoradiographic Study VCR-treated cells were recultured in fresh medium after washing. At 24 hours postrelease from drug, tritiated thymidine (specific activity 2 Ci/mol, ICN Radiochemicals) was introduced into the cultures at a final concentration of 3 ~Ci/ml. Chromosome preparations were made at 18 or 24 hours thereafter. Cells were fixed in a 1 : 1 mixture of acetic acid and methanol and then in a 3 : 1 mixture. The preparations were stained with Giemsa (Merck). Suitable metaphase figures were photographed. After they were destained with methanol, the slides were covered with Sakura NR-M2 emulsion film (Konishiroku Photo Ind. Co. Ltd.) and allowed to expose for 2 weeks. After developing, the slides were restained with Giemsa. The cells previously photographed were rephotographed.
RESULTS AND DISCUSSION
Partial Endoreduplication A total of 278 and 1,021 cells containing diplochromosomes were examined from three sets of the control and VCR-treated cultures, respectively. The frequency of such cells harvested at four time periods was 0.1%-0.3% in control and 1.1%-1.3% in VCR-treated cultures. No metaphase with a mixture of mono and diplochromosomes was observed in the control cultures, whereas 124 cells (12%/ in VCR-treated ones had metaphase with two kinds of chromosomes. The 124 metaphases containing two kinds of chromosomes were classified into three types in the FPG staining pattern (Fig. 1). In type I, all chromatids were darkly stained with Giemsa (Fig. la), indicating that both mono- and diplochromosomes incorporated BrdU once. In type II, two chromatids of each monochromosome and the two inner chromatids of each diplochromosome were stained darkly, while the two outer chromatids of diplochromosomes were pale (Fig. lb), indicating that only diplochromosomes underwent two rounds of DNA replication. In type III, each chromosome of both mono- and diplochromosomes consisted of two different kinds of chromatids, one unifilarly substituted with BrdU and the other bifilarly substituted (Fig. lc), indicating that both mono- and diplochromosomes incorporated BrdU twice. Table 1 shows the distribution of FPG staining pattern of the abnormal metaphases at each harvest time. More than 95% of the metaphases with two kinds of chromosomes were of type II staining pattern. The following two additional experiments were carried out to confirm the partial endoreduplication cycle. First, in order to know the distribution of original strands, BrdU was introduced into the cultures 12 hours before VCR treatment. All metaphases containing two kinds of well-condensed chromosomes showed the type IV FPG staining pattern: namely, one chromatid out of two and four chromatids in each mono- and diplochromosome, respectively, was stained with Giemsa's stain (Fig. ld). Second, in order to trace the timing of monochromosome replication, ]3H]-thymidine was introduced into the cultures during the last 18 or 24 hours before the harvest because a pulse treatment with [3H]-thymidine for less than 12 hours before the harvest failed to label partially endoreduplicated cells. The late replicating regions [11, 18-22] of both mono- and diplochromosomes were labeled. These data from the additional experiments suggest that endoreduplication started on some chromatids of VCR-induced polyploids and the remaining chromatids replicated once during diplochromosome formation. In fact, analysis of 28 well-spread, partially endoreduplicated metaphases revealed that the mean number of total chromosomes and of diplochromosomes found in each metaphase were 37.9 (range 29-48) and 10 (range
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Table 1
T h e D i s t r i b u t i o n of F P G - s t a i n i n g Patterns of 124 Cells C o n t a i n i n g both M o n o c h r o m o s o m e s and D i p l o c h r o m o s o m e s at Each Harvest T i m e Hrs after removal of drug
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Partially endoreduplicated metaphases containing chromosomes showing PC(. (a) Monochromosomes show S-PCC. (b) Diplochromosomes show G2-PCC.
2-17), respectiw,qy, indicating that partial endoreduplication had occurred in neartetrallloid cells. Of 119 partially endoreduplicated cells, 31 (:ells had some chromosomes, either mono- or diplochromosomes, with varying degrees of PCC as shown in Fig. 2. These figures suggest that either set of monochromosomes or diplochromosomes differs in the DNA synthesis pattern; monochromosome replication did not finish until the end of diplochromosome replication (Fig. 2a) or it finished during the second DNA synthetic period of endoreduplicated chromosomes (Fig. 2b). In the former case, as an entrance into the S phase is inducible and as the endoreduplication is considered to be twice as long as the arbitrary cell cycle, markedly delayed monochromosome replication was very surprising. However, it is clear that the initiation signal for DNA synthesis activated one sister nucleus of a multinucleate cell and this nucleus formed diplochromosomes, while the other sister nucleus did not respond well to the stimulation for a long time. This raises the question: What regulates such differences in responsiveness to the stimulation of DNA synthesis? Solving this problem may elucidate the process of partial endoreduplication in detail. Partial Pseudoendoreduplication It is well known that mitotic inhibitors induce aneuploidy probably by mitotic nondisjunction [21-24]. In this case, if the nondisjunction chromosome keeps a centromeric connection, it is considered that the unseparated chromosomes may form diplochromosomes after another round of I)NA synthesis. Indeed, Figures la and 1(: show tile possibility that such a l)henomenon has occurred. In Figure la. 25 chromosomes int:htding seven diph)chromosomes were observed, indit:ating that the cell originated from VCR-induced aneuploidy. Tile existancc of (Jells with the type Ill BrdU-incorporation pattern (:onfirmed indire(:tlv that unseparated (:hronmsonles were induced by the treatment of mitotic inlaibitor. As described above, at least, two mechanisms could produce cells containing both mono- and diplochromosomes in the cultures treated with VCR (Fig. 3). Aneuploidy is formed by VCR-induced nondisjunction (upper line in Fig. 3). A centromeric connection between two sister chromatids leads to nondisjunction of some chromosomes, but a set of chromosomes is enclosed in the same nucleus. If such a nucleus undergoes another DNA synthesis in the presence of BrdU, the type I cell will result.
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Figure 3 Schematic: presentation of replication pattern of partial pseudoendorectuplic:ation (upper line) anct partial endoreduplication (lower line). Dashed lines show BrctU-inc:orporated chrnmatids. Mitotic figures are shown as the FPG staining pattern.
In this mechanism, both mono- and diplochromosomes replicate synchronously. The second mechanism is partial endoreduplication. If one sister nucleus in a VCRinduced multinucleate cell undergoes two rounds of DNA syntheses, whereas the other does so once, the type II cell may be obtained (lower line in Fig. 3).
Segmentally Endoreduplicated Chromosomes in Partially Endoreduplicated Cells Out of 119 partially endoreduplicated cells, 3 cells contained at least one abnormal chromosome (Fig. 4a,c,e). The abnormal chromosome seems to consist of mono- and diplochromosomal segments (Fig. 4b,d,f). Two possible explanations, artifact and segmental endoreduplication, could be considered for the interpretation of these abnormal chromosomes.These chromosomes are actually diplochromosomes, and the monochromosomal segments are formed by two chromatids overlapping during the chromosome preparation. In general, some diplochromosomes, particularly small chromosomes, occasionally show such an overlapping appearance because diplochromosomes are located in three-dimensional fashion within the cell [25]. The overlapped chromatids, indeed, stained darkly and widely as the abnormal segments shown in Fig. 4, comparect with the usual chromatids. However, the chromosome condensation of such overlapped chromatids was similar to that of other normal diplochromosomes or nonoverlapped chromatids. Figure 4a,e show that apparently monnchromosomal segments of the abnormal chromosome condensed more than diplochromosomal segments did. Moreover, the condensation of monochromosomal segments was almost identical to that of conventional chromosomes observed in the same metaphase plate, indicating that the replication and chromosome condensation were coordinated between monochromosomes and monochromosomal segments of the abnormal chromosome in each cell. Therefore, we assumed these chromosomes were formed by delay of DNA replication on some segments of diplochromosomes. Thus, both the process of forming these abnormal chromosomes and their existance in partially endoreduplicated cells are unlikely to be correctly termed "segmental endoreduplication." However, the chromosome itself is segmentally endoreduplicated.
161
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Figure 4 Segmentally endoreduplicated chromosomes. (a,c,e] Partially endoreduplicated metaphases containing a chromosome consisted of both mono- and diplochromosomal segments. (b,d,f) Higher magnification of the abnormal chromosomes (segmentally endoreduplicated chromosomes).
Segmental PCC Segmental PCC is defined as PCC restricted to some segments of a chromosome. Two types of segmental PCC, segmental S-PCC and G2-PCC, were observed at 48 hours postrelease after VCR treatment, although their frequencies were as low as a few cells on one slide (about 2,000 mitotic cells). Chromosomes with segmental S-PCC were easily identified from the remaining chromosomes that were completely organized,
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as shown in Figure 5a. Karyotypic analysis of the cell shown in Figure 5a revealed pulverization of about four fifths of the short arm of X chromosome with slight organization at terminal segments (Fig. 5b). The reason why the replicated terminal segments u n d e r c o n d e n s e and the reason why the lower end of unreplicated segment attaches to chromatid with bifilarly substituted with BrdU are unknown. Figure 5 shows another example of segmental S-PCC of X chromosome. These are very similar to chromosomes presented by Zur Hausen [9] in the sense of u n d e r c o n d e n s a t i o n of some segments in one chromosome. However, in this case, u n d e r c o n d e n s a t i o n was restricted to both long arms of chromosome 1 while in our case, u n d e r c o n d e n s a t i o n occurred on one of the X chromosomes. Two major mechanisms which have been proposed for PCC in the multinucleate cells, a disturbance in the initiation of DNA synthesis (2, 3) or a disturbance in the progression of DNA synthesis (4), may be available for segmental PCC. In addition, it is also possible that these localized PCCs could be provoked by some abnormalities at the replication site, which consists of DNA strands and protein structures. Figure 6 shows an example of segmental G2-PCC. In Figure 6a, a large submetacentric c h r o m o s o m e undercondenses at terminal segments. Karyotypic analysis revealed that the abnormal chromosome was chromosome 1 (Fig. 6b). Figure 6c shows a large ring c h r o m o s o m e with u n d e r c o n d e n s e d segments of about one third. The u n d e r c o n d e n s a t i o n of some segments may occur by two possible mechanisms; inhibition of chromosome condensation or reflection of delay of DNA replication. The first mechanism, inhibition of chromosome condensation, is well documented: several chemical agents that are incorporated into the chromosome [10-15] or that bind to DNA strand [16, 17] have been found to inhibit chromosome condensation. In these cases, the segmental extension occurs in the specific regions. Late-replicating regions are u n d e r c o n d e n s e d in the first-round metaphase of diploid cells after short
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Figure 5 Chromosomes with segmental S-PC'C. (a) Hypotetraploid cell contained a chromosome with incompletely organized segments. (b) A partial karyotype of the cells shown in Figure 5a revealing that one X chromosome has pulverized segments at short arm. (c) A partial karyotype showing one X chromosome with pulw;rized segments.
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Figure 6 Chromosomes with segmental G2-PCC. {a) Hypotetraploid cell contained a chromosome with undercondensed segments. (b) A partial karyotype of the cell shown in Fig. 6a revealing that distal segments of short arm of chromosome 1 undercondensed. (c) A part of metaphase containing large-ring chromosome with undercondensed segments. treatment (during late S phase) with BrdU [10-12] or with 5-azacytidine [13-15[. Centromeric [16, 17] or G-C rich [14] heterochromatins are involved by Hoechst 33258 or 5-azacytidine, respectively. The second mechanism for producing undercondensation of some segments of chromosome is segmental G2-PCC, which reflects the disturbance of DNA synthesis. Although the effect of long treatment with BrdU was not c o m p l e t e l y negligible, we assume these chromosomes were formed by the second m e c h a n i s m because the abnormal chromosomes shown in Figure 6 contain both early and late replicating regions and segments are not restricted to any specific regions. Comments
The term partial pseudoendoreduplicotion has been used to explain the mechanism for formation of triradial chromosomes [8], i.e., after chromatid breakage, some segments attach to the sister chromatid, then replicate and form a triradial chromosome. However, as the aberration occurs in segments of chromosomes, and Drets et al. [26] used the term selective segmental endoreduplication for their symmetrical triradials, segmental p s e u d o e n d o r e d u p l i c a t i o n may be a more suitable term for a triradial chromosome than a partial one. At present, the disturbance in regulation of DNA synthesis has been mainly investigated in multinucleate cells or cells with micronucleus, and it is generalized that PCC is a result of mitoti(: asynchr(my in these (:ells. However, the rare abnormalities restricted to a part of (:hromosome, such as segmental e n d o r e d u p l i c a t i o n and segmental PCC, suggest that a similar disturbance of DNA synthesis o(:(:urs within the nucleus. This further indicates that a disturbance at each step in regulation of DNA synthesis results in many different types of (:hromosomal aberration. Although the abnormal cells described here were rarely observed, they indicate that some minor abnormality occurred during either mitosis or DNA replication after VCR treatment. Also it is likely that such minor abnormalities are more easily detected in late-culture period than in early ones. The biologic effect of these delayed changes of the drug, primary or secondary, is unknown. However, these minor changes may
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p r o d u c e a n e u p l o i d cells. Based on this a s s u m p t i o n and the fact that various p h y s i c a l and c h e m i c a l c a r c i n o g e n s i n d u c e p o l y p l o i d y and m u l t i n u c l e a t e cells, it is suggested that these a b n o r m a l i t i e s may be i n v o l v e d to establish c h r o m o s o m e aberrations of c a n c e r c e l l s - - i n particular, the c h a n g e of the g e n o m e number. Further studies are n e e d e d to e l u c i d a t e the possibility.
Authors wish to thank Dr. T. M. Kou, Department of Molecular Pathology, and Dr. J. C. Liang. Department of Genetics, M. D. Anderson Hospital and Tumor Institute, for critical reading of this manuscript.
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