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[CANCER RESEARCH 36, 2235-2240, July 1976]
Karyotypes of Vasoformative Sarcomas Arising from BALB/3T3 Cells Attached to Polycarbonate Plates I
Michihiro C. Yoshida, Motomichi Sasaki, Noritoshi Takeichi, and Charles W. Boone
Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo, Japan [M. C. Y., M. S.], and Cell Biology Section, Laboratory of Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20014 [N. T., C. W. B.]
SUMMARY
Four vasoformative sarcoma in vivo tumor lines arising from the s.c. implantat ion of 3 • 104 BALB/3T3 cells at- tached to a 1- x 5- x 10-mm polycarbonate platelet were shown to be quite similar in chromosome const i tut ion to the parental 3T3 line. All tumors contained the same marker chromosomes that were present in the parent BALB/3T3 cells. The f indings conclusively showed that the tumors had derived from BALB/3T3 cells and not from host cells or from the in vivo fusion of the inoculated 3T3 cells with host cells. Each tumor line had a unique karyotype that was markedly more homogeneous than that of the parental BALB/3T3 cells, strongly suggesting that each tumor represented a separate clone. An M1 marker chromosome was consist- ently present in each of the four tumor lines but was present in only about 50% of the parent BALB/3T3 cells; it therefore appeared to be a dist inct and stable feature of the tumors.
INTRODUCTION
The BALB/3T3 line, which exhibits the propert ies of marked postconf luence inhibi t ion of cell division (8, 17), anchorage dependence (16), and high serum requirement (19), has been widely used in many laboratories as a repre- sentative nontumor igenic line that can be compared with tumorigenic sublines transformed with various agents (1, 13, 14, 18). Earlier studies (4, 5) demonstrated that the s.c. implantation of less than 3 x 104 BALB/3T3 cells attached to 3-mm glass beads or 1- x 5- x 10-mm polycarbonate plate- lets into syngeneic mice produced tumors, presumably be- cause their anchorage dependence was accommodated in vivo during the initial period of tumor development. In this paper, we have used the quinacrine banding technique to analyze the chromosomes of 4 vasoformative sarcomas arising from s.c. implants of BALB/3T3 cells attached to polycarbonate platelets. The results provide conclusive evi- dence that the tumors did indeed arise from the BALB/3T3 cells, and they support the previous conclusion (5) that each tumor is monoclonal .
MATERIALS AND METHODS
Cell Line. Cline A31 BALB/3T3 cells were obtained from the American Type Culture Collection, Rockvil le, Md., and
, Supported in part by grants for Cancer Research from the Ministry of Education, Science and Culture, Japan.
Received February 5 1976; accepted March 30, 1976.
maintained in Eagle's minimal essential medium supple- mented with 10% calf serum.
Tumors. As described in detail previously (5), 4 in vivo tumor lines, P3/1, P3/2, P3/3, and P3/4, were derived from 4 tumors that arose from the s.c. implantat ion of the A31 BALB/3T3 cells attached to 1- x 5- 10-mm polycarbonate platelets into BALB/c mice.
Karyotypes. Solid tumors removed from animals were minced with scissors in a 0.075 M KCI solut ion containing colchicine, 0.5 /~g/ml, and dissociated by pipet ing. The resulting single-cell suspension was processed for air- drying preparat ions as described previously (22). Slides were stained with Giemsa or quinacrine mustard combined with 33258 Hoechst by the method of Yoshida et al. (21). Karyotypes were arranged according to the standard mouse karyotype (7, 12). The BALB/3T3 cells were processed with essentially the same methods as above.
RESULTS
Karyotype of Parental A31 BALB/3T3 Line. Chromosome numbers of the parental A31 BALB/3T3 line in 50 cells examined varied from 68 to 78 with a mode of 75. Ten karyotypes banded after quinacr ine f luorescence staining, such as the one shown in Fig. 1, are summarized in Table 1. As seen in Table 1, the numbers of each normal chromo- some appeared to be inconsistent from one cell to another except chromosomes 2 and 16, of which 3 copies per cell were present. The X chromosome was usually present in duplicate or tr ipl icate. No Y chromosome was detectable. Nine different chromosomes normal ly not present in mouse metaphases were identified. The origin of these marker chromosomes is il lustrated in Fig. 2. The number of markers ranged from 8 to 15. As shown in Table 1, the BALB/3T3 cells usually lacked 1 or 2 of each marker except M2, M3, M7, and M8, which occurred commonly in all cells. Cells containing every marker chromosome were infrequent (less than 8%).
Karyotypes of Tumors. The number of chromosomes in each line was close to that found in the parental BALB/3T3 cells, but their distr ibut ion was less disperse than those of the parental cells. Each line ranged from 69 to 75 (mode, 73) for P3/1, from 69 to 75 (mode, 72) for P3/2, from 71 to 77 (mode, 75) for P3/3, and from 71 to 78 (mode, 76) for P3/4. The number of intact chromosomes varied from 55 to 69. As shown in Table 2, each line was characterized by its own distinctive chromosome consti tut ion. Marker chromosomes present in tumors were identical with those found in the parental cells (Fig. 3). The number of marker chromosomes
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ranged from 9 to 18 in each tumor line. Each line had a dist inct ive set of markers (Table 2). In addit ion, several marker chromosomes were consistent ly present in all lines: M1, M2, M3, M7, and M8. Line P3/4 acquired a new large biarmed chromosome which resulted from centric fusion of No. 1. Since there was no such biarmed chromosome in the parental 3T3 cells, the 1/1 isochromosome must have oc- curred dur ing tumor progression in vivo. The chromosome const i tut ion in tumor lines appeared to be defini tely more homogeneous than that of the parental 3T3 cells. The distri- but ion of the number of copies per cell of each chromo- some appeared to be more frequently in the tr iploid range than that of the parental 3T3 cells.
D I S C U S S I O N
The similar i ty in karyotype pattern and marker chromo- somes between cells of the A31 BALB/3T3 line and cells of the tumors that arose from implants of BALB/3T3 cells attached to plastic substrates proves conclusively that the tumors did indeed originate from the BALB/3T3 cells and not f rom host cells or from in vivo fusion between the implanted cells and host cells. Every cell of the 4 tumor lines contained the M1 marker chromosome, in contrast to its presence in only about 50% of the parent cells; the M1 chromosome therefore appeared to be a dist inct and stable feature of the tumor lines.
The cells from each tumor had a unique chromosomal complement with a relatively high degree of homogeneity, in contrast to the cells of the parent BALB/3T3 line, which had a relatively heterogeneous chromosome complement simi lar to most commonly used heteroploid lines [the 2 mouse lines MSWBS and RAG, for example, have a remark- ably high degree of heterogeneity associated with the con- t inuous and relatively rapid structural rearrangements (9)]. The fact that each of the 4 tumors possessed a unique karyotype of relatively high homogenei ty supports the con-
clusion reported previously (5) that BALB/3T3 cells may be considered highly preneoplastic and that, when a mono- layer is implanted in vivo, one of the cells in the populat ion eventually undergoes spontaneous transformat ion to the condi t ion of anchorage independence and forms a mono- clonal tumor.
In studies on mal ignant cell t ransformat ion, malignancy or nonmal ignancy has been considered to be related to the gene balance associated with particular chromosomes (3, 6, 10, 11). This balance hypothesis would precisely predict an altered ratio betwen 2 chromosomes. We tabulated the total number of copies per cell of both normal and marker chromosomes in the nontumor igenic parental BALB/3T3 cells and the derived tumor cells. Chromosomes in excess of the 4 expected in a tetraploid cell were Nos. 1,3, 4, 6, 9, 15, 17, 18, and 19 in the BALB/3T3 cells. The tumor cells also contained these excess chromosomes except for No. 9. The fo l lowing chromosomes were consistent ly deficient in both the parental and tumor cells: Nos. 2, 16, and X. Thus, no evidence of a chromosome imbalance in the tumor cells relative to the parental 3T3 cells was seen, although the imbalance hypothesis may not be applied to such heteroge- neous, already imbalanced karyotypes as those in preneo- plastic 3T3 cells and sarcomas originating f rom them.
One tumor line, P3/4, acquired an isochromosome of the 1/1 centric fusion. Since there was no such chromo- some in the parental 3T3 cells, this homology association must have been formed during in vivo progression. The P3/4 was a line that was positive only for endogenous oncornaviruses and reverse transcriptase (5). Whether the format ion of the 1/1 centric fusion is directly related to the presence of endogenous viruses cannot be assessed for the moment, because homology association in chromosomes is frequently observed in mouse cell lines (2, 15, 20).
R E F E R E N C E S
1. Aaronson, A. S., and Todaro, G. J. Basis for the Acquisition of Malignant Potential by Mouse Cells Cultivated in Vitro. Science, 162: 1024-1026,
Table 1 Distribution of the number of copies per cell of chromosomes in BALB/3T3 cell line
No. of cop- Normal chromosome Marker chromosome ies/ cell 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 1 2 3 4 5 6 7 8 m
0 5 4 1 5 2 1 4 4 5 4 9 7 4 6 3 6 2 3 4 1 1 4 2 1 3 4 3 3 1 4 2 7 1 1 1 1 2 3 4 3 5 10 1 1 3 4 4 5 6 4 2 4 4 5 4 10 5 4 4 3 1 1 2 4 2 2 1 6 4 2 3 2 3 8 2 3 2 1 4 1 3 1 5 3 1 1 4 1 1 1 1
Table 2 The modal values for individual chromosomes in 4 tumor lines
No. of Normal chromosome Marker chromosome chromo- somes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 1 2 3 4 5 6 7 8 m 1/1 a
P3/1 73 3 3 3 2 2 3 4 4 3 2 4 3 2 3 3 3 4 2 3 2 1 3 1 1 1 1 4 1 2 P3/2 72 5 3 3 1 4 4 2 3 3 3 4 2 3 2 5 3 4 3 3 3 1 2 1 1 2 1 2 P3/3 75 3 3 3 3 3 3 3 4 4 4 4 2 3 3 5 3 4 3 4 2 1 3 1 2 2 1 1 P3/4 76 3 3 3 3 3 4 3 3 4 4 4 3 3 3 4 3 4 4 4 2 1 1 1 1 1 1 1 1 1
a Isochromosome of No. 1.
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Fig. 1. Karyotype of A31 BALB/3T3. Fig. 2. Marker chromosomes compared to normal chromosomes from the same cells illustrating the possible derivation of the markers. M1, translocation
t(12;17), with break points at 12A1 and 17E5. Centromeric heterochromatin of the No. 12 chromosome was lost. M2, No. 4 with additional bright band of unknown origin on the distal end. M3, No. 3 with additional material near centromere by duplication of a segment or by insertion of a fragment of unknown origin. M4, translocation t(18;?). M5, translocation t(1 ;15), with break points at 1D and 15F. M6, M7, and M8, origin unknown, m, minute.
Fig. 3. Karyotype from tumor line P3/2.
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1976;36:2235-2240. Cancer Res Michihiro C. Yoshida, Motomichi Sasaki, Noritoshi Takeichi, et al. BALB/3T3 Cells Attached to Polycarbonate PlatesKaryotypes of Vasoformative Sarcomas Arising from
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