[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 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. D. B. Stoltz, H. F. Stich, and D. S. Yohn 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- OSS CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association