Chromosome Damage and Polyploidization Induced in Human Peripheral Leukocytes in Vivo and in Vitro with Nitrogen Mustard, 6-Mercaptopurine, and A-649

[CANCER RESEARCH 26 Part 1, 2437-2443, December 19661

CARLOS E. NASJLETI AND HERBERT H. SPENCER

Dental Research Section (C.E.N.) and Hematology Section (H.H.S.), Veterans Administration Hospital, and University of Michigan School of Dentistry (C.E.N.) and Medical Center (Simpson Memorial Institute) (H.H.S.), Ann Arbor, Michigan

Summary onstrated, utilizing streptonigrin or cyclophosphamide (15). Similar chromosomal changes have been induced by chloram- Chromosomal analysLs of peripheral leukocytes in patients being treated with nitrogen mustard, 6-mercaptopurine, and A- phenicol in WBC cultures (C. E. Nasjleti and H. H. Spencer, unpublished data). 649 demonstrated an increase in i>olyploidy, including cells show Although it is now apparent that certain chemotherapeutic ing endoreduplication. With nitrogen mustard, up to 15% of cells cultured were polyploids; with 6-mercaptopurine, 9% poly- agents induce cytogenetic changes in human leukocytes, studies ploidy was evident; anrl when A-649 was used, up to 14% poly- showing these chromosome changes are not numerous. There fore, probes in this direction may help to reveal the biologic ploidy was seen. Chromosomal damage was evident with an effects of chemical agents u]Â»nhuman cells and, in particular, increase in chmmatid breaks and fragmentation. Dicentric may show the frequency in which morphologic changes and chromosomes were the most common rearrangement found. chromosome polyploidization occur. This paper describes the These findings were not present in pretreatment cultures. When structural chromosome aberrations and polyploidization that were leukocytes from normal donors were incubated in vitro with these induced by HN2, 6-MP, and A-649 on human WBC in vivo and 3 agents, similar changes of jjolyploidization and structural ab in vitro. normalities were produced. Care must be taken in the evaluation of chromosomal changes in patients undergoing active treatment. Materials and Methods

Introduction Cytogenetic Studies In Vivo In a recent presentation from this laboratory it was demon Studies were obtained on 5 patients with metastatic carcinoma. strated that radiation from radioactive 131I,wCo,and X-rays has Cases No. 1 and No. 2 had bronchogenic carcinoma and were similar qualitative effects on human chromosomes in vivo and treated with HN2. Case No. 3 had metastatic hypernephroma and in vitro as measured in short-term leukocyte cultures (17). Chro was treated with 6-MP. Case No. 4 had neurofibroma-sarcoma mosomal damage which characterized these effects was of 2 types. and Case No. 5 had metastatic adenocarcinoma of the prostate One type of damage was apparent in the genetic material, gland; both of these were treated with A-649. For these studies, consisting of chromosomal breakage, inversions, deletions, leukocytes from the peripheral blood were cultured by a modifica dicentric forms, ring chromosomes, and other chromosomal tion of the method of Moorhead et al. (14). Samples of venous rearrangements. The 2nd type showed damage in the mitotic blood were obtained before, during, and after chemotherapy in apparatus of the cells, as evidenced by the induction of |X)ly- these patients. As controls, leukocyte cultures from 10normal men ploidy and endoreduplication (P & E)1 of chromosomes. were also examined for chromosome damage and polyploidization. Previously, we described these "radiomimetic" effects in human leukocytes, both in vivo and in vitro, induced with N ,N'-b\s- Cytogenetic Studies In Vitro (3-bromopropionyl) piperazine, an antitumor agent known as A-8103 (16). In leukocyte cultures, mitotic inhibition and Leukocytes from the peripheral blood of 3 healthy human chromosomal damage have been induced with mitomycin (18); donors were cultured for 100 hr instead of the 72 hr used in the in and chromosome breakage and ]>olyploidization have been dem- vivo work. Samples of 12-15 ml of venous blood were obtained from each donor and transferred to sterile vacuum tubes con taining 0.2 ml of heparin solution (1000 units/ml). The vacuum 1The following abbreviations are used: P & E, polyploidy and tubes were placed at an angle of 60Â°and allowed to stand at endoreduplication; A-8103, .Â¡V,.(V-bis-(3-bromopropioiiyl) pipera room teni]>eraturc (approximately 27Â°C)for1 hr. From each tube zine; HN2, nitrogen mustard; 6-MP, 6-mercaptopurine; and A-649, which is NSC 38270, an invest igational antitumor agent duplicate aliquots of 2 ml of supernatant plasma containing obtained through the Midwest Cooperative Chemotherapy Group. viable leukocytes were placed in culture bottles with 8 ml of Received April 4, 1966; accepted June 17, 1966. culture medium 199 (Difco), 700 units of penicillin G, and 0.7 mg

DECEMBER 1966 2437

of streptomycin. To each aliquot 0.5 ml of phytohemagglutinin aberrations was recorded. Final analysis and karyotypes were pre M (Difco) was added. AH aliquots were then incubated at 37Â°C pared according to the Denver System (6). in an atmosphere of 5% carbon dioxide and 95 % air. After 48 hr of incubation the 6 aliquots were taken from the incubator. To Preparation of Chemical Solutions each of 3 aliquots, 1 of the 3 chemotherapeutic agents was added Nitrogen mastard [mechlorethamine hydrochloride (Mus- in concentrations previously tested to induce maximal chromo targen)] was dissolved in distilled water and used immediately some damage without being lethal to the cells. The concentrations after dissolving. 6-Mercaptopurine was dissolved in normal saline used are shown in Tables 2 and 3. The remaining 3 aliquots were by heating in a bath at 85Â°Cfor 10 min. Stock solutions of 6-MP handled exactly like the treated cultures, but did not receive the were stable for months in the refrigerator. A-649, an antibiotic of chemical agents. Following both the actual and the sham chemi unknown empirical formula, was dissolved in ethyl acetate (1 cal treatment, the 6 aliquots were reincubated for an additional mg/ml), and dilutions were made with sterile normal saline. The 48 hr. Four hr before completion, 0.1 ml of Colcemid (Ciba) in a small amount of ethyl acetate present in the cultures was shown concentration of 0.01 mg/ml was added to each culture. At the to be without effect on the chromosomes. end of 100 hr, these cultures were ready for harvesting, fixation, and chromosome spreading. For this, cells were washed twice with Hank's solution, hypotonically treated with distilled water, Results then fixed in a mixture of methyl alcohol and glacial acetic acid Chromosome karyotypes from the 10 individuals of the control (3:1). Drops of this hazy sus|>ension of fixed cells were placed on group were normal in number as well as in morphology. The only tilted microscope slides and allowed to spread. Under these condi variations seen were those generally attributed to artifacts arising tions the cells rupture, allowing the chromosomes to disperse so during preparations. Of 1000 cells counted and analyzed for that they are individually recognizable. Slides were prepared from chromosome damage, 93% had 46 chromosomes, 5% had missing each aliquot, and when jiossible, a minimum of 100 metaphases or in excess 1 or 2 chromosomes, and 2% had chromosome breaks. were analyzed from each culture. The number of chromosome Similarly, the chromosomal patterns in cultured WBC taken from

TABLE 1 CASES,CHEMOTHERAPEDTICAGENTS,ANDCYTOGENETICDAMAGE

(%)Breaks311758441382734139Trans-ABNORMALITIES RELATION CELLSCOUNTED10043025010030010016042018010030016050120100CHROMOSOME CASE12345TREATMENT"HN2HN26-MPA-649A-649CULTURES'TOTREATMENT*B, =Â±46C4973674472754138n=92R"116402yi6/3.032384Â»-92EÂ«1970640320T1265 locations0134010606407221011n

1D,

5A, 4B,

lD, 3A, 1B,

2D,7A,

2B,

1D,3A,

2B,

1D, 2A, 2TOTAL â€¢HN2,nitrogen mustard; 6-MP, 6-mercaptopurine; and A-649,which is NSC 38270,an investigational antitumor agent. 6B, before treatment; D, during; A, after. Number indicates chromosome studies performed. c Indicates metaphases missing or having in excess 1 or 2 chromosomes. 4 Indicates tetraploid metaphases with random chromosome distribution. ' Indicates tetraploid endoreduplicated metaphases with typical diplochromosomes. 1 In each instance includes 1 octoploid metaphase.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Chromosome Damage and Polyploidization in Human Leukocytex the 5 cancer patients before the beginning of therapy did not TABLE 3 differ from the pattern seen in the control group (Table 1). How SUMMARYOF STRUCTURALCHROMOSOMEABERRATIONSIN ever, when using HN2, 6-MP, and A-649 in vivo and in vitro, a CONTROLSAND CULTURES TREATED WITH considerable frequency of structural chromosome aberrations as CHEMOTHERAPEUTICAGENTS well as P & E of chromosomes resulted (Tables 1-3). IN VITRO

Types of chromosome morphologic changes are summarized in (%)Fragments(breaks)4231113Translocations Tables 1 and 3, with an attempt at classification. The changes TREATMENTControlsHN2 CELLSCOUNTED600200200200ABERRATIONS range from very small, hardly visible, acentric fragments to gross and chromatid changes in which chromosome components are engaged in complex exchanges0Hi86Totalinjury4391919 rearrangements (Fig. 3). Simple chromatid breaks were evident in these cultures, as were isochromatid breaks and associated dis placed acentric fragments (Figs. 1, 2, 5). The most common re (0.02)6-MP arrangement was that of dicentric chromosomes (Figs, 1,5), and (0.01)A-649 (0.015)TOTAL it should be [jointed out that, in the analyzed karyotypes of cells containing dicentrics, it would apj>ear that these chromosomes 0 HN2, nitrogen mustard; 6-MP, 6-mercaptopurine; and were formed by the fusion of 2 nonhomologous chromosomes. A-649, which is NSC 38270, an invest Â¡gattonaiaiititumor agent. Tricentric chromosomes were also seen (Fig. 2). Ring chromo somes and typical quadriradial rearrangement of chromosomes incubated with HX2 and 6-MP showed a higher percentage of were observed as well. endoreduplicated metaphases than did the cells treated with Although the following was not investigated or scored for the A-649. Both in vivo and in vitro, tetraploidy was the most common present work, it was noted that the 3 comi>ounds under study are form of polyploidy (Figs. 5, 6), but octoploidy and cells with a capable of inducing or enhancing secondary constrictions in the higher number of chromosomes were also seen in these cultures. chromosomes. These constrictions appeared as short segments of It was noted that these octoploid cells were found with chromo under-stained chromatin of a hazy or puffy nature, and were seen somes in random distribution (Fig. 7) and in quadruplochromo- most frequently in chromosomes Nos. 1, 2, 4, and probably 9. somes (Fig. 8). An example of these is shown in Fig. 4. It should be mentioned, It is interesting that in the in vivo studies, although chromo however, that constrictions, breaks, dicentrics, and other types some aberrations in these patients persisted for some time fol of rearrangements were present in diploid as well as in jxilyploid lowing therapy, a gradual decrease in the number of aberrations metaphases. was noted. In no case were we able to detect chromosomal damage In addition to structural chromosomal aberrations, numerical 3 months after treatment was stopped. Most cells containing variations of chromosomes were also evident. When patients were chromosome aberrations seemed to lose their proliferative capac treated with HN2, up to 15% of cells counted showed i>oly- ity. This was evident from the observation that, in cultures ob ploidization; with 6-MP, 9% polyploidization was evident; and tained 2-3 months following therapy in some of these patients, when using A-649, up to 14% polyploidization was seen. These the large majority of their leukocyte metaphases were charac figures include the jwlyploid metaphases showing random dis terized by a diploid chromosome complement without apparent tribution of chromosomes as well as those showing endoreduplica- chromosomal changes. tion. Similar results were obtained in vitro, although the cells In addition to the structural chromosome disturbances and chromosome polyploidization, other anomalies were disclosed TABLE 2 which seem worthwhile to rejwrt. In microscopic scanning of SUMMARYOF CHROMOSOMECOUNT DISTRIBUTION IN CONTROLS plates from the in vivo and in vitro treated cultures the presence of AND CULTURES TREATED WITH CHEMOTHERAPEUTIC giant cells was evident, especially in HX2 preparations. Super- AGENTS IN VITRO fragmentation of genetic material could also be detected in these plates. It was clear that chromosomes had undergone excessive (%)It TREATMENT(eg/ml)Â«ControlsHN2CELLSCOUNTED600200200200DISTRIBUTION breakage, and fragments showing various degrees of abnormal - Â±46*61098H-46e93707781H- 92tLdÃŒV48Â»=92EÂ«014W3condensation were scattered in the cytoplasm. Koller (10), study ing the effects of HN2 on the cellular morphology of Walker carcinoma cells, characterized this nucleotoxic effect of excessive (0.02)6-MP fragmentation of chromosomes as the result of gross metabolic (0.01)A-649 disturbance induced in the cell by the alkylating agent. (0.015)TOTAL

" HN2, nitrogen mustard; 6-MP, 6-mercaptopurine; and A-649, Discussion which is NSC 38270, an investigational antitumor agent. The findings of structural chromosomal aberrations and ]>oly- '' Indicates metaphases missing or having in excess 1 or 2 chro ploidization in our studies are not surprising. It is well known mosomes. ' Indicates normal diploid metaphases. that HN2 is a radiomimetic substance, since investigations have d Indicates tetraploid metaphases with random chromosome disclosed that the cytologie effects, particularly the injuries to the chromosome material, are very similar to those induced by distribution. ' Indicates tetraploid endoreduplicated metaphases with irradiation (10). Conen and Lansky reported the case of a patient typical diplochromosomes. who showed structural chromosome abnormalities in the cells 1 In each instance includes 1 octoploid metaphase. cultured from peripheral blood after HX2 therapy for malignant

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Carlos E. Xasjleti and Herbert H. Spencer disease (4). Similarly, chromosome damage was reported follow in the damaging process of 6-MP, (6) that following this damage ing cyclophoBphamide (1) or 6-azauridine (5) therapy. there were changes in the mitotic apparatus of the leukocytes In our studies, although many different types of structural with production of polyploid cells, and (r) that some if not all of aberrations were induced with HX2, 6-MP, and A-649 chromo the polyploidization induced in treated WBC occurred through somal breakage and formation of dicentric chromosomes were endoreduplication. In this regard, Hell and Baker (2), in a study among the most common. Chromosome breaks or fragments were of the ratios of tetraploid cells to endoreduplicated cells sampled easily detectable in both in vivo and in vitro plates. It was noted following irradiation, have suggested that tetraploidy was pro that broken chromosomes can behave in 2 different ways: (a) duced by the initial induction of endoreduplication. P & E of they may remain in a fragmented state, or (6) the fragments may chromosomes have been demonstrated in cultured human leu reunite in a new chromosome combination. These chromosomal kocytes with a number of agents, including treatment with A- rearrangements did not follow any specific pattern. Dicentrics 8103, a piperazine derivative (16); /3-mercaptoethanol; ÃŸ- and a few tricentric chromosomes were seen in diploid as well as mercaptopyruvate; and 0-mercaptoethylamine (8). Furthermore, in polyploid mitoses in our treated preparations. It has been exposing colchicine (23) to plant cells and kinetin (9) to animal postulated that the formation of a permanent bridge by a di cells has produced polyploidization of chromosomes. centric chromosome results in cell death because such nuclei in It is apparent from the preceding references and the present subsequent division give rise to abnormal polypolar figures and investigation that one may well expect P & E of chromosomes as giant cells (19). These dicentric chromosomes were commonly well as structural chromosome damage in human leukocytes when observed in our fixed preparations when leukopenia occurred exposed to ohemotherapeutic agents. However, all chemothera- following therapy. Dicentrics were not observed in untreated peutic agents may not induce these chromosomal abnormalities, human leukocytes in our study, although these chromosomes and if changes are produced, different molecular mechanisms may have been observed in euploid human cells (20) and rat cells (11). be at fault. It remains to be established if the administration of Nowell, using mitomycin (18), and Jackson and Lindahl- other chemotherapeutic agents will produce similar chromosomal Kiessling, with sulfhydryl compounds (8), have shown separate aberrations. effects on chromosomal material on one hand, and on the mitotic Our cytogenetie studies of human leukocytes in vivoand in vitro apparatus on the other hand, by treating leukocytes in cultures indicate that HN2, 6-MP, and A-649 induce structural aberra at various time intervals. It is jxistulated (17) that certain muta- tions in addition to P & E of chromosomes. The 5 patients genie agents might damage chromosomes and in addition might examined by us constitute too small a group for any general produce changes in the mitotic process of the cells. Thus, the induction of chromosome polyploidization may indicate mitotic conclusions. Nevertheless, in view of our results, including the damage. in vitro work, the interpretation of cytogenetie changes in pa As noted, we use the term ]x>lyploidization to designate poly- tients receiving active therapy must be reserved. ploidy or endoreduplication (P & E) of chromosomes. Studies indicate that endoreduplicated cells represent a form of )>oly- Acknowledgments ploidy. Jackson and Killander (7), in /3-mercaptoethanol-treated 6-day cultures, observed that the amount of DNA synthesized Thanks are due to Dr. Donald idearle of Burroughs Wellcome was increased in proportion to the degree of polyploidy seen in anil Co., Tuckahoe, New York, for supplying 6-MP; and to Dr. cytologie examinations. DNA measurements obtained by micro- William T. Bradner, of Bristol Laboratories, Syracuse, New York, for supplying A-649. We also wish to acknowledge the technical spectrophotometric studies were in agreement with cytogenetie assistance of Mrs. Jean Simons, Mrs. Virginia Ilartog, and Mr. determination of ploidy, in that tetraploid and octoploid mitoses, Richard Shaw; and the photographic work of Mr. Robert Mc- including endoreduplicated mitoses, contained amounts of DNA Knight and Mr. Stanley Nance. proportional to chromosome number. Endoreduplication was first desrrilx-d by Levan (12) on Allium cepa root cortex as a form of endomitosis and as a process in which internal doubling of ReferenceÂ» chromosomes occurs singly and in multiples. In mouse ascites 1. Arrighi, F. E., Hsu, T. C., and Bergsagel, D. E. Chromosome tumors, Levan and Hauschka (13) found cells in mitotic stages Damage in Murine and Human Cells Following Cytoxan showing groups of 2, 4, or more chromosomes, indicating the Therapy. Texas Rept. Biol. Med., Ã•Q:545-49,1962. doubling of chromosome number during previous interphases. 2. Bell, A. G., and Baker, D. G. Irradiation-Induced Chromo Endoreduplication has been described in 2 cases of acute leu some Aberrations in Normal Human Leukocytes in Cultures. kemia (3, 21). However, Sandberg et al. (22), studying chromo Can. J. Genet. Cytol., 4: 340-51, 1962. somal changes in 75 patients with acute leukemia have stated 3. Bottura, C., and Ferrari, I. Endoreduplication in Acute that the relationship of chromosomal changes to acute leukemia- Leukemia. Blood, el: 207-12, 19(53. is not interpretable at present. 4. Conen, P. E., and Lansky, G. S. Chromosome Damage During Nitrogen Mustard Therapy. Brit. Med. J., Â£:1055-57,1901. The tetraploid metaphase of Fig. 5 is highly interesting. It was found in a culture from the patient treated with 6-MP and has a 5. Elves, M. W., Buttoo, A. S., and Wilkinson, J. F. Chromosome Changes Caused by 6-Azauridine During Treatment of Acute unique structure. It contains 2 identical paired dicentric chromo Myeloblastic Leukaemia. Ibid., 1: 156-59, 1963. somes and 2 sets of chromosome breaks. In addition, one can ob 6. Human Chromosome Study Group. A Proposed Standard serve other paired chromosomes as seen in diplochromosomes of System of Nomenclature of Human Mitotic Chromosomes. J. tetraploid endoreduplicated cells.These observations may indicate Heredity, 61: 213-21, 1960. that (o) the structural chromosome aberrations occurred early 7. Jackson, J. F., and Killander, D. DNA Synthesis in Phy-

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tohemagglutinin-Stimulated Human Leukocyte Cultures and Endoreduplication Induced In Vivo and In Vitro in Human Treated with 0-Mercaptoethanol. Exptl. Cell Res., S3: 459-67, Leukocytes with Ar,Ar'-Bis-(3-bromopropionyl) PiperÃ /ine 1964. (A-8103). Cancer Res., 25: 275-85, 1965. 8. Jackson, J. F., and Lindahl-Kiessling, K. Action of Sulphydrvl 17 â€”â€”â€”.Polyploidization and Aberration ot Human Chromo Compounds on Human Leukocyte Mitosis In Vitro. Ibid., 34-' somes Induced In Vitro and In Vivo with Ionizing Radiations. 515-24, 1964. J. NucÃ.Med., 7: 159-76, 1966. 9. Kevin, S. P., Witkus, E. R., and Berger, C. A. Effects of 18. Nowell, P. C. Mitotic Inhibition and Chromosome Damage Kinetin on Cell Division in Trilurus Viridescens. Ibid., 41: by Mitomycin in Human Leukocyte Cultures. Exptl. Cell Res., 259-64, 1966. SS: 445-49, 1964. 10. Koller, P. C. Comparative Effects of Alkylating Agents on 19. Oster, I. Radiation Effects on Genetic Systems. In: Proceed Cellular Morphology. Ann. N.Y. Acad. Sci., 68: 783-801, 1958. ings of the Conference on Research on the Radiotherapy of 11. â€” â€”. Dicentric Chromosomes in Rat Tumor Induced by Cancer, Madison, Wisconsin, June 16-18, 1960, pp. 45-50. Aromatic Nitrogen Mustard. In: Symposium on Chromosome New York: American Cancer Society, Inc., 1961. Breakage, 1952. Heredity, 6(suppl.): 197-213, 1953. 20. Puck, T. T. Action of Radiation on Mammalian Cells. III. 12. Levan, A. Cytological Phenomena Connected with the Root Relationship Between Reproductive Death and Induction of Swelling Caused by Growth Substances. Ibid., 25: 87-96, 1939. Chromosome Anomalies by X-Irradiation of Euploid Human 13. Levan, A., and Hauschka, T. S. Endomitotic Reduplication Cells In Vitro. Proc. Nati. Acad. Sei. U. S., 44: 772-80, 1958. Mechanisms in Ascites Tumors of the Mouse. J. Nati. Cancer 21. Reisman, L. E., Zuelzer, W. W., and Milani, M. Endore lust., 14: 1-20, 1953. duplication in A Patient with Acute Monocytic Leukemia. 14. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. Lancet, 2: 1038-39, 1963. M., and Hungerford, I). A. Chromosome Preparations of 22. Sandberg, A. A., Ishihara, T., Kikuchi, Y., and Crosswhite, Leukocytes Cultured from Human Peripheral Blood. Exptl. L. H. Chromosomal Differences Among The Acute Leukemias. Cell Res., 20: 613-16, 1960. Ann. N. Y. Acad. Sci., 113: 663-716, 1964. 15. Nasjleti, C. E., and Spencer, H. H. Chromosome Polyploidiza 23. Van't Hof, J. Comparative Cell Population Kinetics of Tri- tion in Human Leukocyte Cultures Treated with Strepto- tiated Thymidine Labeled Diploid and Colchicine-Induced nigrin and Cyclophosphamide. Cancer, in press. Tetraploid Cells in the Same Tissue of Piswn. Exptl. Cell 16. Nasjleti, C. E., Waiden, J. M., and Spencer, H. H. Polyploidy Res., 41: 274-88, 1966.

FIGS. 1-8. Metaphases of 72-hr cultures of peripheral leukocytes from patients treated with HN2, 6-MP, and A-649. Acetic orcein stain. FIG. 1. B, Chromosomal breakage; D, dicentric chromosome. X 1600. FIG. 2. Tetraploid-level mitosis. B, Chromosomal breakage; D, dicentric chromosomes; T, tricentric chromosome. X 1400. FIG. 3. Note complex rearrangement which resulted from chromatid breakage and exchange. X 1600. FIG. 4. Secondary constrictions are indicated by arrows. X 1600. FIG. 5. Tetraploid-level mitosis with chromosomes randomly distributed. However, note paired chromosomes characteristic of endoreduplication (arrows). B, Chromosomal breakage; D, dicentric chromosomes. X 1400. FIG. 6. Tetraploid endoreduplicated mitosis with typical diplochromosomes. X 1400. FIG. 7. Octoploid mitosis with chromosomes randomly distributed. X 1200. FIG. 8. Octoploid endoreduplicated mitosis with quadruplochromosomes. X 1200.

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2442 CANCER RESEARCH VOL. 26

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Chromosome Damage and Polyploidization Induced in Human Peripheral Leukocytes in Vivo and in Vitro with Nitrogen Mustard, 6-Mercaptopurine, and A-649

Carlos E. Nasjleti and Herbert H. Spencer

Cancer Res 1966;26:2437-2443.

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