Model for the Formation of Double Minutes from Prematurely

[CANCER RESEARCH 49. 6731-6737. December 1. 1989] Model for the Formation of Double Minutes from Prematurely Condensed Chromosomes of Replicating Micronuclei in Drug-treated Chinese Hamster Ovary Cells Undergoing DNA Amplification1

Subrata Sen,2 Walter N. Hittelman, Larry D. Teeter, and M. Tien Kuo Divisions of Laboratory Medicine. Hematopathology Program [S. S.J, Medicine fH'. A/. //./. and Pathology fL. D. T., M. T. K.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT The absence of centromeres in DM may result in their unequal segregation during mitosis and, therefore, numerical variations Double minutes (DM) have been associated with gene amplification in in DM among daughter cells. drug-resistant cells and tumor cells. However, the mechanisms by which DM and HSR have been shown to contain amplified genes DM are formed have not been elucidated. \Ye present here a model to in drug-resistant cells. DM-containing drug-resistant cells are describe a possible mechanism of DM formation based on the observa tions made in two independent early drug-selected multidrug-resistant usually unstable. When these cells are grown in the absence of cell lines and from in vitro somatic cell fusion experiments between drug, the resistance rapidly decreases concomitant with the loss synchronized S- and \I-phase cells. The multidrug-resistant cell lines of DM in these cells. Interconversion between DM and HSR contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic has been suggested (6, 10, 11). In tumor cells, amplified onco- analyses of cells at early stages of selection revealed the presence of a genes in DM have also been noted (3). These amplified onco- number of micronuclei in a subpopulation of these cells. These micronu genes may render growth advantage to these cells. clei were often asynchronous in their progression through the cell cycle. Despite the significant roles of DM in drug-resistant cells As a result, premature condensation of micronuclear chromatin was often and tumor cells, the detailed mechanisms for DM formation observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes dis have not been elucidated. Recently, Carroll et al. (12, 13) have plays a striking resemblance to DM structures. These DM-like structures reported the presence of submicroscopic autonomously repli are linked by replicating DNA as revealed by DNA labeling experiments. cating circular molecules in cells with amplified genes and Somatic cell hybrids between S- and M-phase cells when grown in vitro proposed that DMs are formed from these extrachromosomal demonstrated that S-phase prematurely condensed chromatin indeed DNA molecules. gives rise to extra chromosomal structures in the successive cell genera In this paper, a model is presented to describe the possible tions. It is hypothesized that distinct DM-like structures may arise from route of DM formation. Our model is primarily based on the partially replicated and prematurely condensed S-phase chromosomes Cytogenetic observations made in two early passage drug-resist following their liberation as extra chromosomal entities after replication ant cell lines which contain DM and an amplified mdr (P- and/or recombination in the succeeding division cycle(s). The enrichment glycoprotein) gene and also display DM-like structures (11). In for DM containing specific genes in drug-resistant cells may result from a significant number of these cells which were being continu the subsequent drug selections. ously treated with drug, thin fibrillar structures were distinctly visible around the DM. These structures resembled the appear INTRODUCTION ance of S-PCC. These structures were virtually absent from the DM3 and expanded chromosomal segments known as HSR parental CHO line from which the drug-resistant cells were selected. Chromosome labeling experiments of the drug-treated are two distinct chromosomal abnormalities that have been cells revealed these structures to indeed represent S-PCC. Cell described in a number of cell lines either selected for resistance to cytotoxic drugs or derived from tumors (1-4). Typically, hybrids resulting from fusion of S- and M-phase cells subse DM are 0.3- to 0.5-Â¿imchromatin particles (5), but their size quently showed that S-PCC structures can give rise to extra chromosomal DM-like entities. The paper describes studies varies from limits of light microscopic resolution to that of a human G group chromosome (6). In addition to this size which led to the development of a model suggesting involve ment of the S-PCC phenomenon in the formation of DMs. heterogeneity, the number of DM per cell has been found to vary greatly among different cells, i.e., ranging from a few (7) to more than 1000 per cell (8). Cytogenetic observations of MATERIALS AND METHODS squashed preparations from metaphase cells revealed clusters of DM, suggesting close spatial association of DM structures Cell Culture. The CHO cell line and its drug-resistant variants were maintained as monolayer cultures at 37Â°Cin 5% COj in air in Dulbec- in the nucleus. Ultrastructural studies suggested that DM are co's modified Eagle's medium (Gibco Laboratories, Grand Island, NY) chromosomal segments but lack functional centromeres (5, 9). supplemented with 10% fetal bovine serum (Hazelton, Denver, PA) Received 12/9/87; revised 6/7/89. 8/28/89: accepted 9/1/89. containing 0.1% neomycin (Pharma-Tek). Procedures for selection of The costs of publication of this article were defrayed in part by the payment these drug-resistant mutants by vinblastine and Adriamycin have been of page charges. This article must therefore be hereby marked advertisement in accordance with 18 L'.S.C. Section 1734 solely to indicate this fact. described earlier (11). Two of the low levels of drug-resistant mutants 1Supported in part by grants from the Robert A. Welch Foundation (G 831 selected with 1.0-Mg/ml concentrations of vinblastine (VBR 1.0) and to M. T. K.) and the NIH (GM28573 and CA43621 to M. T. K: CA2793I and Adriamycin (ADR 1.0) at early passages (within 10 to 15 passages of 45746 to W. N. H.). ! To whom requests for reprints should be addressed, at the University of their first exposure to this concentration of the drugs) were used for Texas M. D. Anderson Cancer Center. Division of Laboratory Medicine. Box 72, our study. 1515 Holcombe Blvd.. Houston. TX 77030. Cytogenetic Procedures. The frequency of cells with mitotic anoma 3The abbreviations used are: DM. double minutes: HSR. homogenously stain lies was analyzed in squash preparations. Cells were trypsinized, pel ing regions; dThd. thymidine; PCC. prematurely condensed chromosomes; S- leted by centrifugation, and resuspended gently in a hypotonie solution PCC. S-phase prematurely condensed chromosomes; CHO. Chinese hamster ovan cells; VBR, vinblastine resistant cells; ADR. Adriamycin resistant cells; (medium:water, 3:1) for 10 min. Cells were collected by centrifugation, BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline: S/M hybrid, S- fixed with methanohacetic acid (3:1, v/v), and stained with 2% ace- and M-phase-fused cells: M/M hybrid. M- and M-phase-fused cells. toorcein stain. Squash preparations of these cell pellets were made on 6731

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. MECHANISM OF DM FORMATION clean slides after covering with 22-mm2 coverslips. ratories, and was used in consultation with Dr. Joshua Epstein of the DNA Labeling Analyses. To detect labeled multinucleated cells, Department of Hematology at the University of Texas-M. D. Anderson semiconfluent monolayer cultures were labeled with 6 nC\/m\ of ['H|- Cancer Center. Replicative behavior of the extrachromosomal struc dThd (specific activity. 6.7 Ci/mmol; ICN, Irvine, CA) for 20 min, and tures in the hybrids was also studied following autoradiography of the the cells were directly fixed in methanohacetic acid (3:1, v/v). Air-dried slides made from cells grown in |'H]dThd after fusion. slides were then processed for autoradiography. In the case of labeling DNA associated with DM, semiconfluent cultures were similarly incubated with 6 ^Ci/ml of |'H]dThd for 20 RESULTS min, washed twice with regular medium containing unlabeled dThd (0.1 HIM), and again incubated for 10 more min at 37Â°C.Colcemid Cytogenetic observations in two cell lines, VBR1.0 and (0.16 ^M) was included in the medium throughout the entire 30-min ADR 1.0, have led us to propose the present model for double period of labeling and washing the cells. Cells were harvested, and air- minute formation. These cell lines were being selected to survive dried slides were then processed in one of the following ways, (a) One in media containing low concentrations of vinblastine and Adri- set of slides was first autoradiographed to reveal the grain distribution amycin, respectively. These two cell lines display the multidrug on metaphase cells. The silver grains were removed, and the same cells were examined for DM-like structures around the same area, (b) An resistance phenotype and contain amplified mdr (P-glycopro- other set of slides was first stained with Giemsa (5% in 0.01 M NaPO4, tein) gene (H). A significant subpopulation (20 to 30%) of cells pH 6.8) to observe the DM-like structures in the metaphase cells and in both VBR1.0 and ADR 1.0 displayed the presence of DM (or then autoradiographed to see if replicating DNA could be localized in DM-like structures) in their metaphase spreads. The number of the vicinity of DM. DM varied from 0 to about 50 in both cell lines. Autoradiography was performed with Kodak NTB-2 nuclear track In many instances, these extrachromosomal structures emulsion (diluted 1:2 in water) and developed in D19-B solution. The showed resemblance with S-PCC. For these structures to be exposure time was about 10 days at 4Â°C.To remove silver grains from products of PCC, it was hypothesized that heterophasic nuclei autoradiographs, the slides were rinsed in water for about 5 to 10 min, in micronucleated cells had to occur following exposure of the treated with 0.22 M potassium ferricyanide solution for 3 min, trans cells to the drugs and that premature condensation of the ferred to 1.2 M sodium thiosulfate for 3 to 5 min, and then rinsed in chromatin from the micronucleus lagging behind the primary three changes of water for 1 min each. Slides were then stained with Giemsa or ethidium bromide (5 ng/m\ in citrate-phosphate buffer, pH nucleus in its entry into mitosis had taken place. We, therefore, 5.5) and observed under light or fluorescence microscope. sought to score the frequency of cells with abnormal metaphase Cell Synchronization, Cell Fusion, and Preparation of Chromosome plates and micronuclei. Slides. Mitotic CHO cells were obtained by gentle shake off of subcon- Analyses of the squash preparations of directly fixed cells fluent cultures treated with 0.05 Mg/ml of Colcemid for 3.5 h. Only revealed that a significantly higher proportion of both VBR 1.0 preparations with mitotic indices greater than 95% were utilized for all and ADR1.0 lines display mitotic anomalies compared with the subsequent experiments. Synchronized S-phase cells were obtained by parental CHO line. The anomalies identified included lagging washing mitotic cells free of Colcemid and allowing the cells to divide chromosomes during anaphase separation, multipolar mitoses and proceed into the next cell cycle. Preliminary experiments showed (Fig. \A), and resulting cells with micronuclei (Fig. IB). The that the labeling index of these populations reached 95 to 97% by 7 h. frequency of such cells was found to be about 3.2% in VBR 1.0, Thus, for cell fusion experiments, cells synchronized to and released 3.1% in ADR 1.0, and 0.6% in the CHO line (Table 1). from mitosis were incubated for 7 h with 5 Mg/ml of BrdUrd, prior to In order to determine whether DM-like structures were re fusion with mitotic CHO cells. The procedure for cell fusion has been lated to S-PCC, in cells being continuously exposed to drug previously described (14). Briefly, about equal numbers of S- and M- phase cells were washed twice separately and twice after mixing in during early stages of selection, we performed DNA labeling Hanks' balanced salt solution. The cell pellet was resuspended in 0.5 experiments with ADR 1.0 and VBR 1.0 cells. The cells were ml of medium without serum containing UV-inactivated Sendai virus briefly labeled with ['HjdThd, harvested, exposed to hypotonie and incubated at 4Â°Cfor 15 min to allow agglutination. Fifty jul of 20 solution, fixed, and then autoradiographed. Twenty-one HIMMgCl2 and 50 n\ of 0.5 ng/m\ of Colcemid were then added, and ADR 1.0 and 24 VBR 1.0 metaphase cells that contained clusters the fusion mixture was incubated at 37Â°Cfor 45 min. At the end of the of silver grains were photographed. Examples of such labeled incubation time, an aliquot of cells was withdrawn for chromosome cells are shown in Fig. l, D and F. After removal of silver preparation, and the rest was replated in equal aliquots of 10 ml of culture medium with or without 0.5 Â¿iCi/mlof['HjdThd (6.7 Ci/mmol; grains, the slides were restained with Geimsa or Ethidium Bromide, and the same metaphase plates were reexamined. We ICN) for different time intervals. The cells were harvested at 24 h post found that, in all the cells analyzed, DM-like structures were plating and onwards. The first harvest immediately after fusion is present in the same areas as the replicating DNA (Fig. 1, C and referred to as time zero, and all others are indicated by the number of hours of their growth in culture before being harvested for slide prepa E). A detailed examination of the distribution of silver grains ration. Air-dried slides were prepared following treatment of cells with revealed that the replicating DNA is situated around but not a hypotonie solution of 0.075 M KC1 and their fixation in metha- overlying the condensed chromatin resembling DM bodies. nol:glacial acetic acid. This correlation strongly suggested a relationship between S- Detection of BrdUrd-substituted Chromosomes. The differential stain phase PCC and DM-like structures seen in these cells. ing method of Perry and Wolff (15) and a modified immunological It was further hypothesized that the replicating DNA in S- detection method of Speit and Vogel (16) were used to monitor the fate PCC containing metaphase cells must be very labile and, as this of BrdUrd-labeled chromatids of the S-phase parent cell in the hybrids. S-phase PCC-containing metaphase cell progresses through the For immunostaining, slides were treated with methanohNaOH (0.1 M) following cell cycles, chromosomal deletions at the site of (5:2, v/v) for 5 to 10 min, to denature the DNA, rinsed in PBS, treated with Triton X-100 for 5 min, and rinsed in PBS again. Slides were then replicating DNA may disconnect DM bodies from the replicat blocked in PBS-containing 5% serum and incubated with a 1:200 ing DNA and result in the formation of extrachromosomal dilution of a mouse monoclonal anti-BrdUrd antibody (IU-4) for 1 h. entities, such as DM (Fig. 1C) (see below). In this context, one would predict that not all the DM or DM-like structures seen Following another rinse and block, slides were stained with fiuorescein isothiocyanate-conjugated rabbit antimouse IgG. Slides were counter- in every metaphase plates would be associated with replicating stained with 4,6-diamidino-2-phenylindole. The anti-BrdUrd antibody, DNA. Indeed, in the above experiment, when metaphase plates IU-4, was a kind gift from Dr. Joe Gray, Lawrence Livermore Labo were chosen for examination, on the basis of the presence of 6732

Fig. 1. Composite figures showing the se quence of events leading to the formation of DM-like structures in VBR1.0 and ADR 1.0 cells. A. an abnormal metaphase cell showing multipolar movement of chromosomes and lagging chromosomes during segregation, li. micronuclei (arrows) containing replicating DNA associated with unlabeled nucleus in the same cell. C to F, autoradiographic analyses of replicating DNA associated with S-PCC. Cells were labeled according to the procedures de scribed in "Materials and Methods." Autora diographic pictures were taken (D, F), and the same plates were degrained and restained with Giemsa (C) and ethidium bromide (E). Note that pulverized chromatin resembling DM-like structures is seen in fand Â£whichare adjacent to the silver grains seen in /) and /. G, a metaphase plate of VBRI.O showing DM (ar row).

Table 1 Frequency of cells with micronuclei and mitotic anomalies scored in of cells may become aneuploid due to chromosome nondisjunc- squash preparations tion and form multipolar metaphase plates (Stage 2), leading withmicronucleiand to the formation of micronuclei in the subsequent cell cycle cellsscored814698776Cellsmitoticanomaly5::24Frequency(%)0.63.153.09(Stage 3). Occasionally, micronuclei are not in the same phase CelltypeCHOVBRI.OADR of cell cycle as the main nucleus in the polykaryon. When such a cell carrying heterophasic nuclei progresses to metaphase, 1.0Total condensation of chromosomes results in the formation of PCC from micronuclei. In the case of S-PCC, the micronuclear chromatin forms a pulverized configuration connected by dif DM, only 4 of 25 ADR 1.0 and 5 of 28 VBRI.O metaphase fuse chromatin fibers. Most often this diffuse chromatin con plates were found to contain replicating DNA around the DM. tains replicating DNA (Stage 4). When this metaphase cell On the basis of these observations, the following model is proceeds to anaphase, incompletely replicated condensed chro proposed for the formation of DM (see drawing in Fig. 2). mosomes may give rise to DM-like structures (Stage 5). The When animal cells are treated with cytotoxic agents, a fraction chromatin bodies, generated through S-PCC, may eventually 6733

Fig. 2. A proposed model for double min ute formation. Normal cell (Stage 1) exposed to cytotoxic drug resulting in formation of mitotic abnormality in metaphase (Stage 2). When this eell progresses into the following cell cycle, micronuclei (A/A') are formed (Stage Asynchronous 3). Micronuclei and main nucleus (A') may Progression asynchronously progress into metaphase, re sulting in the formation of S-PCC in micron- S-PCC Formation uclcar rinomatiti (Stage 4). Chromosomal deg radation, replication, and/or recombination in S-PCC leads to release of chromosomal bodies like DM (Stage 5).

be established as distinct DM structures following their repli mosomes in one of the parents. Results from these independent cation and/or recombination in the next division cycle. Ampli experiments show that, at 24 h post fusion, about 6% of the fication of their DNA in the population of cells under selection hybrids displayed extra chromosomal fragments. This fre could take place through anomalous segregation of these struc quency was reasonably high, since the frequency of S-PCC in tures in the succeeding cell generations. the original fusion between S-phase and mitotic cells at time To test the validity of this hypothesis for DM formation, zero ranged between 7 and 9%. With extended periods of hybrid cells containing S-phase PCC were generated by fusing growth in the absence of any selective pressure, however, the BrdUrd-labeled S phase cells with M phase cells (S/M hybrids). number of hybrids with such extra chromosomal structures As a control, mitotic cells were fused with mitotic cells (M/M showed a distinct decline. This implies that, under normal growth conditions in cultures, hybrids with extra chromosomal hybrids). The hybrid cells were grown in culture and examined structures are at a selective growth disadvantage compared with at subsequent mitoses. Table 2 illustrates the fate of S-phase prematurely condensed the parental cells. M/M hybrids, on the other hand, exhibit almost no induction of extra chromosomal structures. An oc chromosomes in S/M hybrids and in M/M hybrids. S/M hy casional M/M hybrid with such structure is seen. This is prob brids were distinguished from other homophasic hybrids on the ably a secondary consequence of segregation anomalies taking basis of differential staining of the BrdUrd-substituted chro- place in the tetraploid cells generated after fusion. Having seen the generation of extrachromosomal structures hybridsExperimentExperimentTable 2 Fate of S-PCC in S/M and M/M in the dividing S/M hybrids in culture, the authors were inter of ested in determining how a partially replicated chromosome metaphases of with PCC and/or (i.e., S-phase PCC) replicates in the next cell cycle and, specif metaphases extrachromosomal ically, if, in succeeding cell generations following fusion, these (i =)024384402444024384402444024384602446No. scored125130112128125105101100115110122114130110ion103105103596847No.fragments9853187431196621%7.26.14.42.30.9863.62.40.70.995.85.71.91.4structures first replicated their unreplicated regions or autono 1S/MM/MExperiment mously replicated their replicated segments from the previous cell cycle. To address these issues, the S-phase cells were labeled with BrdUrd immediately prior to fusion, and following fusion, the hybrids were grown either in the absence of the analogue or in some cases in the presence of 'H-labeled thymidine. The BrdUrd-labeled chromatids can be distinguished from unsub- 2S/MM/MExperiment stituted chromatids with the help of immunostaining and/or Hoechst dye-mediated differential staining techniques. The rep licative performance of BrdUrd-substituted chromatids follow ing premature condensation in hybrids could also be monitored due to their differential staining characteristics. Thus, if the BrdUrd-substituted PCC structures were to replicate once or twice in the absence of the analogue and incorporate thymidine, .1S/MM/MTime then they would have either differentially stained or uniformly stained sister chromatids in the ensuing cell generations. Also the spatial and numerical arrangement of sister chromatids would indicate if the previously replicated regions reinitiated replication or not, and if the replicated segments fell apart. In anti-BrdUrd antibody-stained and differentially stained 6734

Fig. 3. Composite figures showing the generation of extra chromosomal structures and their replication in representative S/M phase hybrid cells harvested at 44 to 46 h post fusion in different experiments. A. a metaphasc plate of a hybrid cell showing anti-BrdUrd antibody- stained extrachromosomal structures. The S-phase parent cell chromatin was labeled with BrdUrd prior to fusion that gives rise to these structures. Specificity of the staining is indicated by the distinct differential staining of the two chromatids in the metaphase chromosomes of a cell la beled with BrdUrd as shown on the righi. B. Hoechst fluorescence plus Giemsa-mediated differential staining of the extrachromosomal structures in a hybrid cell derived from BrdUrd-labeled S-phase parent. Differentially stained structures shown with arrows indicate that they have replicated at least once following fusion in the ab sence of BrdUrd. C. a hybrid cell between a mitotic and BrdUrd-labeled S-phase cell when grown in the presence of [3H]dThd shows differential staining of some of the extra chromosomal structures (â€”Â»).O,radioactive precur sor incorporation in them indicating their replication dur ing in vitro growth in culture.

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hybrid cells (Fig. 3, A to C), extra chromosomal structures seen and movements (segregations) might also facilitate breakage of in S/M hybrids at 24 h and later after fusion were derived from the replicating DNA. Furthermore when S-PCC structures S-phase prematurely condensed chromatin. Fig. 3A shows the reinitiate replication in the next cycle and/or undergo recom extrachromosomal structures positively stained with the anti- bination at the chromosomal sites with nicked DNA strands, BrdUrd antibody, verifying their derivation from the S-phase extrachromosomal structures could be generated in the process. parent cell chromatin that was prelabeled with BrdUrd in early Whatever the mechanisms involved, our results presented in S phase prior to fusion. Differential staining of the chromatids Table 2 clearly show that S/M heterophasic cells give rise to a of these structures (Fig. 3B) reinforced this conclusion and higher frequency of DM-like structures in subsequent cell gen indicated further that the prematurely condensed replicated erations than the M/M homophasic cells. These results support segments rereplicated in the subsequent S phase. Moreover, the the idea that S-PCC segregate through cell division cycles, rereplicated regions appeared to become distinct DM-like struc reinitiate DNA synthesis, and fall free. tures. The replicative potential of these structures was further DM are extrachromosomal bodies that may contain ampli confirmed by their ability to incorporate [JH]dThd as shown in fied DNA sequences encoding gene products as targets for the Fig. 3, C and D. The differential staining pattern of the chro actions of specific drugs. The model describing the early steps matids was not equally distinct in all the extra chromosomal in the formation of DM can be extended to accomplish gene structures of S/M hybrids. Whether or not this reflects differ amplification in the following manner. The partially replicated ences in the replication behavior of some of these structures is PCC structures often segregate to one of the daughter cells as not clear. double entities during the first anaphase, making the recipient daughter cell 4n with respect to the replicated segment. Follow ing the next round of replication, these regions of the genome DISCUSSION would become 8n. Further random segregation of the DM-like This report describes a simple model for the origin of DM structures could allow the copy number of their sequences to through PCC of S-phase micronuclei in cells exposed to drugs. gradually increase in subsequent cell generations. The model assumes that micronuclei are initially formed fol The enrichment of specific DNA sequences in DM in drug- lowing mitotic anomalies. Vinblastine and Adriamycin, the two resistant cells might be a result of continuous selection pressure selective drugs studied with our cell lines, despite having differ which favors the growth of cells containing DM harboring ent targets of action inside the cells (e.g., microtubule for specific genes conferring drug resistance. The drug treatment vinblastine and chromosomes for Adriamycin), share the com thus seems to have the dual function of first, initiating partially mon feature of inducing multiple nucleation. Micronuclei and replicated genomes that could serve as substrates for generation mitotic anomalies can be seen not only in vinblastine- and of DM and thereafter selecting cells with extra copies of the Adriamycin-treated cells, but also in animal cells treated with gene which enhances growth and survival. It is conceivable that a number of other cytotoxic agents. Micronuclei-inducing the same model could be applied to the formation of DM in agents need not be mitotic poisons, e.g., Colcemid or Vinca tumor cells containing amplified oncogenes. Several oncogene- alkaloids. In fact, micronuclei formation has long been consid encoded proteins are involved in regulation of cell proliferation, ered an index for standard mutagenicity assay (17). Among the and cells with DM containing these oncogenes may have growth 20 or so known agents that have been shown to induce DM advantages over the others (3). formation or gene amplification in cultured cells, many, if not Multiple mechanisms may be involved in gene amplification all, are capable of inducing micronuclei. Furthermore, if mi in drug-resistant cells. In some instances of drug-selected cell cronuclei formation were an important initial event in the lines and of cells established from tumor samples, DM may production of DM, as proposed in this model, one would predict arise from small circular episomal DNA as proposed by Wahl that DM-containing cells are aneuploid. This has indeed been and his Â«workers (13, 20, 21). In other instances, however, it predominantly seen, and DMs have been observed rarely in is plausible that large DNA segments are liberated from chro normal diploid cells. mosomes to form DM, as proposed here, especially in view of DNA labeling experiments were performed to show that the the fact that, in multidrug-resistant CHO cells, amplification polykaryons that formed in the drug-treated cells often traverse units have been found to be over IO6 base pairs long (22). In the cell cycle in distinct asynchrony. Such asynchrony in drug- fact, the model described in this paper can explain the formation treated polykaryons has been observed previously, and occa of both microscopic and submicroscopic precursors of DM. sional induction of PCC also has been reported (18, 19). The This model does not address the question of how the amplified mechanism of asynchrony among nuclei in the same cell is still DNA is organized in DM. Whether these abnormal chromo not clearly known. somal structures contain DNA rereplicated within one cell The presence of pulverized chromatin of S-phase nuclei as cycle, as has been suggested, remains to be determined (23). an intermediate step in DM formation as described in the present model is consistent with the idea that DM are produced ACKNOWLEDGMENTS through chromosomal deletions. We have proposed that some The authors are thankful to Dian Miller and Gloria Clinkscales for times deletions occur in those regions of the genome which are typing the manuscript and to Phylisha Agbor for technical assistance. induced to prematurely condense while undergoing DNA rep Subrata Sen is thankful to Dr. Sanford A. Stass, Director of the lication. One possibility to explain this phenomenon is that the Hematopathology Program and Dr. Emil J. Freireich, Director of the replicating DNA which is in diffused chromatin configuration Adult Leukemia Research Program in the Institute, for their support and encouragement during the concluding stages of this study, and to is very sensitive to endogenous nucleases. Alternatively, pre his wife, Dr. Pramila Sen, for her help in preparation of this manuscript. mature condensation of chromatin may interfere with the nor mal DNA replication machinery, leaving gaps and unligated REFERENCES DNA strands. As metaphase cells containing S-phase PCC 1. Hamlin. J. L., Milbrandt. J. D.. Heintz. N. H., and Azizkahn. J. D. DNA further proceed through the cell division cycle, chromosome sequence amplification in mammalian cells. Int. Rev. Cytol.. 90: .11-82. structural changes (continuing condensation/decondensation) 1984. 6736

2. Stark. G. R.. and Wahl, G. M. Gene amplification. Annu. Rev. Biochem.. deletion. Mol. Cell Biol.. 8: 1525-1533. 1988. S3.-447-491, 1984. 14. Hittclman. W. N. Premature chromosome condensation for the detection of 3. Stark. G. R. DNA amplification in drug resistant cells and in tumors. Cancer mutagenic activity. In: T. C. Hsu (ed.). Cytogenetic Assays of Environmental Sun.. 5: 1-23. 1986. Mutagen. pp. 353-384. Totwa. NJ: Osman Allanheld. 1982. 4. Schimke, R. T. Gene amplification in cultured cells. J. Biol. Chem.. 263: 15. Perry. P.. and Wolff, S. New Giemsa method for differential staining of sister 5989-5992. 1988. chromatids. Nature (Lend.). 251: 156-158. 1974. 5. Barker. P. E., and Stubblefield. E. Ultrastructure of double minutes from a 16. Speit. G.. and Vogel. W. Detection of bromodeoxyuridine incorporation in human tumor cell line. J. Cell. Biol.. 83: 663-666, 1979. mammalian chromosomes by a bromodeoxyuridine antibody. Chromosoma 6. Cowcll, J. K. Double minutes and homogenously staining regions. Gene (Beri.). 94: 103-106. 1986. amplification in mammalian cells. Annu. Rev. Genet.. 16: 21-59. 1982. 17. Jenssen. D., and Ramel, C. The micronucleus test as part of a short term 7. Morgan. R. T.. Woods. L. K.. Moore, G. E., McGraven, L., Quinn, L. A., mutagenicity test program for the prediction of carcinogenicity evaluated by and Sample. T. V. A human gall bladder carcinoma cell line. In Vitro, 17: 143 agents tested. MutÃ¢t.Res., 75: 191-202, 1980. 503-510, 1981. 18. Stubblefield. E., DNA synthesis and chromosomal morphology of Chinese 8. Levan, G., Mandahl. H., Bregula. U.. Klein, G., and Levan. A. Double hamster cells cultured in media containing jV-deacetyl-A'-methylocolchicine minute chromosomes are not centromeric regions of the host chromosome. (Colcemid). In: J. R. C. Harris (ed.). Cytogenetics of Cells in Culture, pp. Hereditas. 83: 83-90. 1976. 223-248. New York: Academic Press. 1964. 9. Hamkalo. B. A.. Farnham. P. J.. Johnston. R.. and Schimke, R. T. Ultra- 19. Obe, G., and Beek. B. Premature chromosome condensation in micronuclei. structural features of minute chromosomes in a methotrexate resistant mouse In: P. N. Rao. R. T. Johnson, and K. Sperling (eds.). Premature Chromosome 3T3 cell line. Proc. Nati. Acad. Sci. USA, 82: 1126-1130. 1985. Condensation. Applications in Basic. Clinical, and Mutation Research, pp. 10. Martinsson, T., Dahlof. B., Weltergren, Y., Leffler. H., and Levan, G. 113-130. New York: Academic Press, 1982. Pleiotropic drug resistance and gene amplification in a SEWA mouse tumor 20. Von Hoff. D. D.. Needham-Vandevanter. D. R., Yucel, J., Windle, B. E., cell line. Exp. Cell. Res.. 75Â«:382-394. 1985. and Wahl. G. M. Amplified human myc oncogenes localized to replicating 11. Sen, S., Teeter. L. D,. and Kuo, T. Specific gene amplification associated submicroscopic circular DNA molecules. Proc. Nati. Acad. Sci. USA, 85: with consistent chromosomal abnormality in independently established mul- 4804-4808. 1988. tidrug resistant Chinese hamster ovary cells. Chromosoma (Beri.), 95: 117- 21. Wahl, G. M. The importance of circular DNA in mammalian gene amplifi 125, 1987. cation. Cancer Res., 49: 1333-1340, 1989. 12. Carroll. S. M.. Gaudray, P., De Rose, M. L., Emery, J. F., Meinkoth. J. L., 22. DeBruijn. M. L.. Vander Blick. A. M.. Biedler. J. L., and Borst, P. Differ Nakkim, E., Subler. M.. Von Hoff. D. D., and Wahl. G. M. Characterization ential amplification and disproportionate expression of five genes in three of an episome produced in hamster cells that amplify a transfected CAD multidrug resistant Chinese hamster lung cell lines. Mol. Cell Biol.. 6:4712- gene at high frequency. Functional evidence for a mammalian replication 4722. 1986. origin. Mol. Cell Biol.. 7: 1740-1750. 1987. 23. Schimke, R. T., Sherwood, S. W., Hill, A. B.. and Johnson, R. N. Over- 13. Carroll, S. M.. De Rose, M. L., Gaudray. P.. Moore, C. M.. Needham replication and recombination of DNA in higher eukaryotes: potential Vandevanter, D. R.. Van Hoff. D. D., and Wahl, G. M. Double minute consequences and biological implications. Proc. Nati. Acad. Sci. USA, 83: chromosomes can be produced from precursors derived from a chromosomal 2157-2161.1986.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. Model for the Formation of Double Minutes from Prematurely Condensed Chromosomes of Replicating Micronuclei in Drug-treated Chinese Hamster Ovary Cells Undergoing DNA Amplification

Subrata Sen, Walter N. Hittelman, Larry D. Teeter, et al.

Cancer Res 1989;49:6731-6737.

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