Viruses and Mammalian Chromosomes VII. the Persistence

[CANCER RESEARCH 27 Furt l, 587-598, March 1987]

Viruses and Mammalian Chromosomes VII. The Persistence of a Chromosomal Instability in Regenerating, Transplanted, and Cultured Neoplasms Induced by Human Adenovirus Type 12 in Syrian Hamsters1

D. B. STOLTZ, H. F. STICH, AND D. S. YOHN Department of Biology, McMaster University, Hamilton, Ontario, Canada, and Viral Oncology Section, Roswell Park Memorial Institute, Buffalo, New York

SUMMARY questions: Is the chromosome instability an inherent feature of the neoplastic cells; are new karyotypes continuously formed; The incidence of cells with karyotypic abnormalities, such as are particular neoplastic cells characterized by particular chrom pseudodiploidy and aneuploidy, and of cells with chromosome osome aberrations; and do stemlines evolve during tumor pro aberrations, including breaks, fragmentations, and coiling gression? anomalies, was estimated in regenerating, transplanted, and cultured neoplasms induced in Syrian hamsters by human MATERIALS AND METHODS Adenovirus type 12. The various neoplasms studied consisted of karyotypically heterogeneous cell populations. Numerous clones Tumors with particular chromosome complements were present, but Regenerating. Primary tumors, induced by human Adeno stemlines were not a dominant feature of the neoplastic cell virus type 12 in newborn hamsters (32), were allowed to grow for populations examined. A relatively high incidence of chromo 3 weeks, at which time they had reached a diameter of 2-3 cm. some aberration persisted in all neoplasms. A possible relation Eighty % of the tumor was then removed, and the tumor allowed ship between retention of the viral genome or part of it in the to regenerate. Regeneration was typically faster than primary neoplastic cells and the persistence of a chromosome instability and the significance of chromosome aberration in Adenovirus- growth, so that subsequent operations, up to a maximum of 5, on these tumors were performed at 2-week intervals. 12-induced oncogenesis are discussed. Transplanted. All transplanted tumore were originally de rived from a single female tumor-bearing Syrian hamster. Small INTRODUCTION pieces, about 1 cu mm, from actively growing areas of this tumor Primary neoplasms induced by human Adenovirus type 12 in were implanted s.c. in the dorsal area of 3-week-old, newly Syrian hamsters are characterized by karyotypically heteroge weaned inbred hamsters. Conditioning by means of cortisone neous cell populations, by the lack of a predominant aneuploid was found to be unnecessary, and tumor takes of at least 90% stemline, and by a relatively high incidence of chromosomal and were invariably obtained. After 1 month of growth, the tumors mitotic irregularities, including chromatid breaks, gaps, over- were either serially transplanted in the same way or examined contracted or partially uncoiled chromosomes, and chromosome cytologically. fragmentation (32). These neoplastic cells contain virus-associ ated T-antigens (10, 13, 16, 21 29), indicating that at least a Tissue cultures portion of the viral genome persists in these cells and is trans mitted to subsequent progen}'. We have previously speculated Established cultures from tumors of 2 different female ham sters were maintained on 1066 medium, supplemented with 10% (21, 32) on the possibility that the instability of the chromosome inactivated fetal calf serum, and 100 fig,each of penicillin G and complement in the primary neoplasms either is linked to the streptomycin sulfate. The medium was changed ever}' 2 days, presence of the viral genome or a product of it or results from a and the cultures were subeultured twice weekly. Cells prolifer primary action of the virus which, at least in rilro, induces a ated rapidly and there was no tendency for cultures to die off. relatively high incidence of chromosome aberrations in as short a period as 24 hr (21). The present paper deals with the further evolution of Adenovirus-type-12-induced neoplastic cell jwpula- Cytologie Preparations tions. Particular emphasis has been placed on the following Metaphase plates were arrested by colchicine (i.p. injection of 1 ml of 0.1 % colchicine per 100 gm body weight; 2 hr). Actively 1Supported in part by the National Cancer Institute of Canada growing tumor tissue was minced and placed in distilled water and in part by Grant CA-07745from the USPHS. for 2 min, and then fixed in Carnoy's fluid. Tissue was pretreated Received May 31, 1966;accepted October 7, 1966. 15 min in 45% propionic acid and squashed on albumin-coated

MARCH 1967 587

TABLE l Incidence of Karyotypically Abnormal Cells in Adenovirus-type-12-induced Neoplastic Cell Populations of Regenerating, Transplanted, and Cultured Tumors from the Syrian Hamster

karyo- diploids 4- typically ab plates with TumorRegenerating chromosome pseudotetra- normal meta- plates chromosome plates nos. ploids phase plates observed436667659520410713178Metaphaseaberrations" observed43666705273502136578328122 (%)1004770626838459998Pseudo-(%)09.0154.03262551.08Total(%)100568566100100100100100Metaphase(%)28183132422932505754Metaphase

tumorsAH14, (d")AH12,3rd regrowth (9)AH2,4th regrowth )AH4,5th regrowth ( 9 (cf)Transplanted5th regrowth

tumorsTumor ITumor IIITumor VIICultured

tumorsCulture vitroCulture4a, 11 months in vitroCulture46, 11 months in 7, 16 months in vitroAneuploid " Includes chromatid and isochromatid breaks, fragmentation, and double minutes in euploid, pseu" dodiploid, and aneuploid cells.

slides, which were then heated over an alcohol burner. Cover The chromatid and isochromatid breaks may lead to exchanges slips were removed in 50% alcohol, and slides were then air-dried. and thus to the formation of new chromosomes with an abnormal Staining was in 2% orcein, although a few slides prepared by an morphology. Several examples of marker chromosomes, including air-drying technic (22) were stained with a basic fuchsin, methyl- long dicentrics, telocentrics, and submetacentrics, are illustrated green mixture. in Figs. 3, 4, 6,14, 15, and 16. Many of these marker chromosomes Chromosomes of well-spread metaphase plates were classified were observed only once or twice, whereas others appeared in according to Lehman et al. (18), although such a system must be several metaphase plates from a given sample of tumor tissue. regarded as being rather superficial (24). Acentric fragments resulting from chromosome breakage com monly formed micronuclei (Fig. 8). RESULTS Abnormal Chromosome Numbers. All the neoplasms examined were characterized by a relatively high incidence of Chromosome Aberrations. All the regenerating, trans mctaphase plates with aneuploid chromosome numbers. Since planted, and cultured neoplasms examined were characterized by most colchicine-treated metaphase plates were well spread, an a wide spectrum of chromosome aberrations, including breaks, accurate count was possible even in cells with high chromosome fragmentation, and coiling deficiencies. The total incidence of numbers (Fig. 7). The incidences of aneuploid metaphase plates chromosome aberrations for these neoplasms is given in Table 1, in the regenerating, transplanted, and cultured tumors are and the frequency of the most common types of aberrations is summarized in Table 1, and the spread of chromosome numbers shown in Table 2. Of particular interest is the relatively high in the various neoplasms examined is shown in Table 3. With the proportion of metaphase plates having one or more fragmented exception of the neoplastic cells in tissue culture, diploid and chromosomes, whereas the morphology of the remaining ones is near diploid chromosome numbers predominated among the cell apparently normal (Figs. 1, 2). It is likely that the loss of these Imputations of the regenerating and transplanted tumors fragmented chromosomes from a karyotype represents a mech examined. anism by which aneuploid chromosome numbers might originate. Abnormal Karyotypes. Since chromosome counts show only Occasionally, a large part or all of the chromosome complement variations in chromosome number and do not reveal any chromo showed an extreme fragmentation which one might term pul some rearrangements or the presence of marker chromosomes, a verization (Fig. 9). Another peculiarity common to all 3 types detailed analysis of several karyotypes of each of the neoplasms of neoplasms studied, although especially noticeable in the tumor tissue cultures, is the so-called "double minutes" (Fig. 5). Several studied was performed. Particular emphasis was placed on the question whether metaphase plates with diploid chromosome hyperdiploid metaphase plates contained more than 30 such double minutes. These structures are either fragments originating numbers have a normal diploid karyotype. Two typical examples by multiple isochromatid breaks, or they represent real minute are shown in Figs. 14 and 15. Although both karyotypes contain chromosomes having centromeres. Despiralization of chromosome 44 chromosomes, they differ in their marker chromosomes and in arms (Figs. 10, 12), and even of single chromatids (Fig. 11). was the number of chromosomes in Groups 11-15 and 16-19. Both frequently found, especially in the tumor tissue cultures. cells were monosomic for chromosome No. 20. These pseudodip-

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TABLE 2 Freauency of Metaphase Plates with Particular Chromosome Aberrations in Adenovirus-type-12-induced Neoplaslic Cells of Regenerating Transplanted, and Cultured Tumors in the Syrian Hamster

TumorRegenerating fragmenta minutes fragmenta minutes(%)4.740Negligible44.03.0133410BreaksÂ«(%)6.0NegligibleÂ±8Â»fragmenta tion(%)33.45.26.04.01.54.77.27.5Double(%)Negligible2.0Negligible0.92.30.77.04.35.0Breaks"(%)21271426254.46.81611Â±4Â»tion(%)2.36.729165.40.720310Double tion(%)3060Doubleminutes(%)3040

tumorsAH14, (cf)AH12,3rd regrowth )AH2, 4th regrowth ( 9 )AH4, 5th regrowth ( 9 (d")Transplanted5th regrowth

tumorsTumor ITumor IIITumor VIICultured

tumorsCulture vitroCulture4a, 11 months in vitroCulture46, 13 months in 7, 16 months in vitroBreaks"(%)151226302221118.723Â±2Â» " Includes chromatid and isochromatid breaks.

TABLE 3 Chromosome Counts of Adenovirus-type-ie-induced Tumors in the Syrian Hamster

TumorRegenerating

tumorsAH14, (cf)AH 3rd regrowth (9)AH2,12, 4th regrowth (9)AH4,5th regrowth (d")Transplanted5th regrowth

tumorsTumor ITumor IIITumor VIICultured

tumorsCulture vitroCulture4a, 11 months in vitroTumorRegenerating7, 16 months in

tumorsAH14, (d*)AH12,3rd regrowth (9)AH2,4th regrowth )AH4, 5th regrowth ( 9 (d")Transplanted5th regrowth

tumorsTumor ITumor IIITumor VIICultured

tumorsCulture vitroCulture4

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TABLE 4 Karyotypic Variability of Pseudodiploid Cells in Transplanted Tumor III

groupsX2-1-2-11

to5106 to1010+ to1510+1+2+ to198-1-1-1-21-1+1+1202-1-1-1-1-1-1-11-1-1-1-1212+

Normal number of chro ineachmosomes present )Deviationsgroup (9 normal"infrom transplanted tu mor cellNo.:123467891011121314Chromosome telocentricLong submetacentricLong 111 telocentricLong

116 telocentricLong telocentricLong 1+2MarkerLongtelocentricLong telocentricLong submetacentricLong submetacentricLong submetacentricLong submetacentric

Translocations may account for what appears to be a loss of certain chromosomes.

loid karyotypes may contain additional cryptostructural chromo planted Tumor III, 72 out of 124 pseudodiploid metaphase plates some anomalies. The apparently unequal length between the 2 were characterized by a particular karyotype with a long telo homologs of Pair 1 in Fig. 15 might indicate such a cryptostruc centric chromosome, monosomy for No. 20, only 7 chromosomes tural change. Since, however, the detection of small structural in group 16-19, and an extra chromosome in Group 11-15 (Fig. changes is ambiguous, they were not used in the identification of 14). It appears likely that these 72 metaphase plates comprise a pseudodiploidy. The detection of small deletions and transloca stemline. In addition, 24 out of the 124 pseudodiploid metaphase tions which do not change markedly the lengths of a chromosome plates examined from this tumor contained the long telocentric or the position of its centromere is made still more difficult by the marker chromosome, but otherwise differed karyotypically from inability to recognize positively the homologs of most chromo the stemline and among themselves. The}' probably represent somes of the Syrian hamster (18). The pairing of chromosomes as derivatives of the stem cell. shown in Figs. 14-17 must be, therefore, considered as tentative. No predominant stemline was observed in the cultured tumors. Several examples of different pseudodiploid karyotypes in a trans Even metaphase plates with the same chromosome number ex planted tumor are give in Table 4. The incidence of pseudo hibit a great variation in their chromosome complements (Table diploid metaphase plates in the various neoplastic cell iwpulations 5). examined is presented in Table 1. With the exception of regenerating Tumor AH14, all the neo DISCUSSION plasms examined contained some metaphase plates with hypodiploid chromosome numbers. Several of these hypodiploid Predominant stemlines are usually found in primary neoplasms karyotypes were characterized by marker chromosomes or by the or else may evolve in the course of transplantation (4, 8, 11, 14, loss of particular chromosomes. An example of a hypodiploid 23, 31). However, the primary neoplasms induced in Syrian ham karyotype is given in Fig. 16. A clone of hypodiploid cells charac sters by human Adenovirus type 12 (32) and the regenerating, terized by a long telocentric chromosome was observed in Tumor transplanted, and cultured neoplasms studied lacked a predom tissue culture 4o. inant stemline. It is likely that the persistence of a relatively high Most hyperdiploid karyotypes not only have additional chro incidence of chromosome aberrations leads to a continuous forma mosomes but also are characterized by either marker chromosomes tion of new chromosome complements, thus preventing the estab or a rearranged chromosome complement, or both. The karyotype lishment of a stemline. This observation seems to point to an of a cell with 45 chromosomes is illustrated in Fig. 17. inherent instability of the chromosome complement in Adeno Stemlines. Predominant stemlines were apparently absent in virus type-12-induced neoplasms. A comparable chromosome in the regenerating tumors examined. However, each of these stability has been observed in hamster cells transformed by SV40 tumors had several clones of cells with particular marker chro (2, 35). Nevertheless, it would appear erroneous to conclude that mosomes, chromosome rearrangements, and chromosome num karyotypic instability is a characteristic feature of all neoplasms bers. An indication of one or more relatively stable clones has induced by oncogenic viruses. For example, leukemias induced by been found in the transplanted tumors. For example, in trans Friend, Rauscher, Rich, or Gross viruses and those which are

590 CANCER RESEARCH VOL. 27

TABLE 5 (1,5,13, 25, 27, 29). The presence in primary, regenerating, trans Variations in the Number of X, Marker, #20, 21, and 16-19 planted, and cultured Adenovirus-12-induced neoplasms of a Chromosomes virus-associated T-antigen (10, 21) and of particular cytoplasmic fibers which stain with ferri tin-labeled Adeno virus-12 T-anti- variabilityX111222222111122221112222Marker"112111222111211111111111Chromosomeused in analysis of bodies (16) shows that at least part of the viral information must Chromosome number696969696969696969707070707070707072727272727272Chromosomes survive and be consistently transmitted over many cell genera Â«20433-444424-5423444444-534433-444-5ChromosomeÂ«21242222222222222222222222ChromosomesÂ«16-1913-14121312119-1013-141199-10111311121113-149131112-13139-101114Other1tions. It is possible, therefore, that the neoplastic properties of the cell [Â»pulation studied are a consequence either of the pres ence of the viral genome itself or of some product coded or in duced by it. Although the high incidence of chromosome aberrations com mon to all the neoplasms examined can perhaps be best regarded longtel-ocentric as a secondary phenomeon, apparently unnecessary for the maintenance of the neoplastic state, it is nevertheless possible that they may be involved in some initial phase of Adeno virus-12- induced oncogenesis. If it should be proven, for example, that the DNA of an oncogenic virus actually becomes incorporated into the genome of the host, then one could suggest that chromosome breakages, such as are common in hamster cells recently infected with Adenovirus 12 (21), might provide the opjwrtunity for such an integration to occur. It is well known that the "open" ends of broken chromatids have a tendency to associate with the "open" ends of other broken chromatids, leading to such phenomena as recombination, reunion, and translocation (33). If it should be proven that some comparable mechanism of virus-chromosome interaction occurs during the early stages of Adenovirus-12- induced oncogenesis, then the significance of chromosome aberra tion in the transformation of normal cells into malignant ones should be sought in the establishment of this virus genome-cell genome complex. REFERENCES " Long subtelocentric marker chromosomes as shown in Figs. 1. Axelrad, D., Bolton, E. T., and Habel, K. Polyoma Viral 18-21. Genetic Material in a Virus-free Polyoma Induced Tumor. Science, 146: 1466-1468, 1964. spontaneous in AKR mice (36) consist of cells with a stable dip- 2. Cooper, H. L., and Black, P. H. Cytogenetic Studies of 3 Clones Derived from a Permanent Line of Hamster Cells loid or aneuploid chromosome complement and lack an excep Transformed by SV40.J. Cellular Comp. Physiol., 64: 201-220, tionally high incidence of chromosome breaks or fragmentations 1964. (15, 26, 34). 3. Ford, C. E. Selection in Mammalian Cell Populations. In: It is generally thought that different karyotypes convey dif R. J. C. Harris (ed.), Cytogenetics of Cells in Culture, pp. 27- ferent selective advantages to neoplastic and nonneoplastic cell 45. New York: Academic Press, 1964. populations in vitro and in vivo (3, 4, 6, 7, 9, 11, 12, 17, 19, 20, 4. Ford, C. E., Hamerton, J. L., and Mole, R. H. Chromosomal 28, 30). Indeed, the presence of clones and the predominance of Changes in Primary and Transplanted Reticular Neoplasms of the Mouse. J. Cellular Comp. Physiol., 52: 235-269, 1958. pseudodiploid and near-diploid chromosome numbers, especially 5. Gerber, P. Virogenic Hamster Tumor Cells : Induction of Virus in the solid tumors studied, seem to suggest the selective ad Synthesis. Science, 145: 833, 1964. vantages of some karyotypic categories over others. However, the 6. Hauschka, T. S. Cell Population Studies on Mouse Ascites great variety of karyotypes seen in the primary (32), regenerating Tumors. Trans. N. Y. Acad. Sci., 16: 64-73, 1953. transplanted, and cultured Adenovirus-12-induced tumors indi 7. Hauschka, T. S. Correlation of Chromosomal and Physiologic cates that the malignant properties of these tumor cells do not Change in Tumors. J. Cellular Comp. Physiol., 52 (Suppl. 1): depend to any great extent on a particular chromosome comple 197-233, 1958. ment and that any selective forces that may be present are rela 8. Hauschka, T. S. The Chromosomes in Ontogeny and Oncog- tively nonspecific. Furthermore, it can be argued that a wide eny. Cancer Res., 21: 957-974, 1961. spectrum of abnormal karyotypes can persist only where the 9. Hauschka, T. S., Kvenar, B. J., Grinnel, S. T., and Amos, D. B. Immunoselection of Polyploids from Predominantly Dip- maintenance of a neoplastic state is not a function of a particular loid Cell Populations. Ann. N. Y. Acad. Sci., 63: 083-705, karyotype. 1956. What, then, is the source of the high frequency of chromosome 10. Hoggan, M. D., Rowe, P. W., Black, P. H., and Huebner, R. aberrations seen in Adenovirus-type-12-induced neoplastic cells? F. Production of "Tumor Specific" Antigens by Oncogenic There is increasing evidence that the viral genome, or a part of it, Viruses during Acute Cytolytic Infections. Proc. Nati. Acad. persists in cells transformed byDXA-containingoncogenie viruses Sei. U. S., 53: 12-19, 1965.

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11. Hsu, T. C. Chromosomal Evolution in Cell Populations. In 23. Makino, S. The Chromosome Cytology of the Ascites Tumors tern. Rev. Cytol., 12: 69-161, 1961. of Rats, with Special Reference to the Concept of the Stemline 12. Hsu, T. C., and Kellogg, I). S., Jr. Mammalian Chromosomes Cell. Intern. Rev. Cytol., 6: 26-S4, 1957. in Vitro. XII. Experimental Evolution of Cell Populations. J. 24. Patau, K. The Identification of Individual Chromosomes, Nati. Cancer Inst., gfc 1067-1093, 1960. Especially in man. Am. J. Human Genet., 12: 250-276, 1960. 13. Huebner, R. J., Howe, W. P., Turner, H. C., and Lane, W. C. 25. Pope, J. H., and Rowe, W. P. Immunofluorescent Studies of Specific Adenovirus Complement Fixing Antigens in Virus- an Adenovirus 12 Tumor and of Cells Transformed or Infected free Hamster and Kilt Tumors, with Preliminary Observations by Adenoviruses. J. Exptl. Med., ISO: 577-537, 1964. on Similar Antigens in Other Virus-induced Tumors. Proc. 26. Rich, M. A., Tsuchida, R., and Siegler, R. Chromosome Nati. Acad. Sei. U. S., 50: 379-339, 1963. Aberrations: Their Role in Etiology of Murine Leukemia. 14. Joneja, M. G., and Stich, H. F. Chromosomes of Tumor Cells. Science, 146: 252-253, 1964. IV. Cell Population Changes in Thymus, Spleen, and Bone 27. Sabin, A. B., and Koch, M. A. Behavior of Noninfectious SVÂ« Marrow during X-ray Induced Leukemogenesis in C57BL/6J Viral Genome iu Hamster Tumor Cells: Induction of Synthesis Mice. J. Nati. Cancer Inst., S5: 421-434, 1965. of Infectious Virus. Proc. Nati. Acad. Sei. U. S., 50: 407-417, 15. Joneja, M. G. and Stich, H. F. Chromosome Aberrations in 1963. Virus-induced Murine Leukemia. Exptl. Cell Res., Jfl: 148-151, 28. Sachs, L., and Gallily, R. The Chromosomes and Transplant- 1965. ability of Tumors. II. Chromosome Duplication and the Loss 16. Kalnins, V. I., Stich, H. F., and Yohn, D. S. Electron Micro of Strain Specificity in Solid Tumors. J. Nati. Cancer Inst., scopic Localization of Virus Associated Antigens in Human 16: 803-841, 1956. Amnion Cells (AV-3) Infected with Human Adenovirus, Type 29. Smith, K. O., and Melnick, J. L. Adenovirus-like Particles 12. Virology, 28: 751-754, 1966. from Cancers Induced by Adenovirus-12 but Free of Infectious 17. Kaziwara, K. Derivation of Stable Polyploid Sublines from Virus. Science, Â¡45:1190-1192, 1964. Hyperdiploid Ehrlich Ascites Carcinoma. Cancer Res., 14: 30. Stich, H. F. Mosaic Composition of Preneoplastic Losions and 795-801, 1954. Malignant Neoplasma. Exptl. Cell Res., Suppl. 9, pp. 277-235, 18. Lehman, J. M., MacPherson, I., and Moorhead, P. S. Karyo- 1963. type of the Syrian Hamster. J. Nati. Cancer Inst., SI: 639-650, 31. Stich, H. F. Chromosomes and Carcinogenesis. Can. Cancer 1963. Conf., 6: 99-115, 1963. 19. Levan, A. Relation of Chromosome Status to the Origin and 32. Stich, H. F., and Yohn, D. S. Viruses and Mammalian Chromo Progression of Tumors. The Evidence of Chromosome Num somes. IV. Chromosome Aberrations in Adenovirus Type 12 bers. In: Genetics and Cancer, pp. 151-182. Austin: Univ. of Induced Tumors of Syrian Hamsters. J. Nati. Cancer Inst., Austin Press, 1959. 35: 603-615. 20. Levan, A., and Biesele, J. J. Role of Chromosome in Cancero- 33. Swanson, C. P. Cytology and Cytogenetics. Englewood Cliffs, genesis as Studied in Serial Tissue Culture of Mammalian N.J.: Prentice-Hall, 1957. Cells. Ann. N. Y. Acad. Sci., 71: 1022-1053, 1953. 34. Tsuchida, R., and Rich, M. A. Chromosomal Aberrations in 21. MacKinnon, E., Kalnins, V. I., Stich, H. F., and Yohn, D. S. Viral Leukemogenesis. I. Friend and Rauscher Leukemia. J. Viruses and Mammalian Chromosomes. VI. Comparative Nati. Cancer Inst., SS: 33-43, 1964. karyologic and Immunofluorescent Studies on Syrian Hamster 35. Vogt, M., and Dulbecco, R. Steps in the Neoplastic Trans and Human Amnion Cells Infected with Human Adenovirus formation of Hamster Embryo Cells by Polyoma Virus. Proc. type 12. Cancer Res., 26: 612-618, 1966. Nati. Acad. Sei. U. S., 49: 171-179, 1963. 22. MacPherson, I. Characteristics of a Hamster Cell Clone Trans 36. Wakonig, R., and Stich, H. F. Chromosomes in Primary and formed by Polyoma Virus. J. Nati. Cancer Inst., SO: 795-815. Transplanted Leukemias of AKR Mice. J. Nati. Cancer Inst., 1962. 25: 295-305, 1960.

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FIG. 1. Metaphiise plate with several fragmented chromosomes. Cultured Tumor 4a. X 1000. FIG. 2. Metaphase plate with 6o chromosomes, showing 1 fragmented chromosome (arrow). Cultured Tumor 4a. X 2000. FIG. 3. Part of an aneuploid metaphase plate with 4 dicentric chromosomes (arrows). Cultured Tumor 4a. X lÃ¶OO. FIG. 4. Part of an aneuploid metaphase plate with a single, exceptionally long, dicentric chromosome. Transplanted Tumor III. X 4000. FIG. 5. Part of a metaphase plate showing numerous double minutes (D) and a ring chromosome (arrow). Cultured Tumor 7. X 2000. FIG. 6. Part of a hyperdiploid metaphase plate showing a single dicentrio chromosome. Cultured Tumor 4a. X 4000.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. Fio. 7. Aneupluid metaphase plate with 129 chromosomes. Cultured Tumor 4o. X 1.300. Fio. 8. Interphase cell with a single micronucleus (arrow). Cultured Tumor 46. X 1500. FIG. 9. Part of a metaphase plate showing partial pulverization of the chromosome complement. Cultured Tumor 7. X 1500. FIGS. 10, 11. Two examples of coiling anomalies (arrows). Regenerating Tumors. X 4000, X 2000. FIG. 12. Aneupluid metaphase plate showing erosion of several chromosome arms (arrow). Cultured Tumor 7. X 1500. FIG. 13. Part of an aneuploid metaphase plate showing several single minutes (arrow). Cultured Tumor 46. X 4000.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. FIG. 14. Pseudodiploid karyotype: an extra chromosome in the 11â€”15group,1 missing from the 16-19 group, and a long telocentric marker chromosome (A/). Chromosome No. 20 lacks a homolog. Transplanted Tumor III. X 5000. FIG. 15. Pseudodiploid karyotype: a long submetacentric marker chromosome (A/) is present. Chromosome No. 20 lacks a homolog. Transplanted Tumor III. X 5000. FIG. 16. Subdiploid karyotype (39 chromosomes): at least 1 X chromosome is missing, as well as 3 chromosomes in the 16-19 group. Chromosome No. 20 lacks a homolog. There are an extra chromosome No. 21 and a very long submetacentric marker chromosome (A/) with a secondary constriction. Cultured Tumor 4a. X 5000. FIG. 17. Hyperdiploid karyotype (45 chromosomes): 2 extra chromosomes in the 11-15 group, and 1 missing from the 16-19 group. Chromosome No. 20 lacks a homolog. There is a long telocentric marker chromosome (A/). Transplanted Tumor III. X 5000.

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596 CANCER RESEARCH VOL. 27

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. FIGS. 18-21. Metaphase plates showing karyotypic variability within clones found in cultured Tumor 4o. X 2000. FIG. 18. Metaphase plate with 70 chromosomes showing 2 X chromosomes (A"), and 1 long subtelocentric marker (Ã¬l).Eleven telocentric and two No. 21 chromosomes (arrows). FIG. 19. Metaphase plate with 70 chromosomes showing 1 X, 1 long subtelocentric, 11 telocentric, and 2 No. 21 chromosomes. FIG. 20. Metaphase plate with 74 chromosomes showing 2 X, 1 long subtelocentric, 13 telocentric, and 1 No. 21 chromosomes. FIG. 21. Metaphase plate with 73 chromosomes showing 1 X, 1 long subtelocentric, 11 telocentric, and 2 No. 21 chromosomes.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. Viruses and Mammalian Chromosomes. VII. The Persistence of a Chromosomal Instability in Regenerating, Transplanted, and Cultured Neoplasms Induced by Human Adenovirus Type 12 in Syrian Hamsters

D. B. Stoltz, H. F. Stich and D. S. Yohn

Cancer Res 1967;27:587-598.

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