Coincidence of cytogenetic markers in four murine cell lines

Coincidence of Cytogenetic Markers in Four Murine Cell Lines Irene Beatriz Larripa and Sonia Brieux de Salum

Foreign body tumorigenesis was induced by the subcutaneous implantation of a plastic or glass cylinder in BALB/c mice; the inoculation of human neoplastic cells significantly increased the incidence of these anaplastic sarcomas. Of 15 tumors studied, four presented the same markers: one induced with and three without human neoplastic cell inoculation within the foreign body. The markers observed were double minutes (DM), a long acrocentric marker (MLA), and a metacentric marker (MM). The DM are a number of small often tiny chromosomal structures appearing in pairs together with chromosomes of ordinary size. MLA is a long acre° centric derived from a translocation in tandem between chromosomes #1 and #16. MM is due to centric fusion of two chromosomes #10. Numerical anomalies consisted of gains of the same chromosomes types. It is postulated that these coincident findings are related to the foreignbody tumorigenesis.

ABSTRACT:

INTRODUCTION This p a p e r reports the cytogenetic s t u d y of three cell lines d e r i v e d from foreign b o d y i n d u c e d sarcomas of mice. A total of 15 tumors were s t u d i e d for p l o i d y a n d presence of markers. F o u r were selected for further b a n d i n g studies. In a p r e v i o u s p a p e r [1] w e described some cytogenetic finding in the P193 cell line and P193 t u m o r develo p e d in a BALB/c m o u s e carrying a plastic c y l i n d e r containing neoplastic h u m a n material w h i c h had been i m p l a n t e d s u b c u t a n e o u s l y 9 m o n t h s earlier [2]. In a detailed b a n d i n g s t u d y of the r e m a i n i n g three tumors, CS1, CS2, a n d CS3, w e f o u n d the same n o n - r a n d o m c h r o m o s o m a l rearrangements, attributable to foreign b o d y tumorigenesis or to a viral activation a c c o m p a n y i n g t u m o r d e v e l o p m e n t or to both. The m u r i n e cell lines CS1, CS2, a n d CS3 d e r i v e d from three anaplastic sarcomas from three different BALB/c mice carrying e m p t y plastic cylinders, also imp l a n t e d 9 m o n t h s earlier. The c h r o m o s o m e abnormalities d e t e r m i n e d by G- and C-banding techniques consistently f o u n d were (a) the c h r o m o s o m e aberration referred to as d o u b l e m i n u t e s (DM), m i n u t e c h r o m a t i n bodies (MCB), m i n u t e chromosomes, or m i c r o c h r o m o s o m e s , that consisted of a n u m b e r of small, often tiny, c h r o m o s o m a l structures a p p e a r i n g in pairs together w i t h c h r o m o s o m e s of o r d i n a r y size, and (b) a long marker acrocentric (MLA): t(lq;16q), a t a n d e m translocation of c h r o m o s o m e s # 1 and #16. The presence of d m and MLA c o i n c i d e d in almost all karyotypes.

From the Institute de Investigaciones Hematol6gicas de la Academia Nacional de Medicina, Buenos Aires, Argentina. Address requests for reprints to Dr. LB. Larripa, Institute de Investigaciones Hemato16gicas de la Academia Nacional de Medicina, Av. Las Heros 3092 (1425), Cap. Fed., Buenos Aires, Argentina. Received November 1, 1980; accepted December 12, 1980.

169 © Elsevier North Holland, Inc., 1981 52 Vanderbilt Ave., New York, NY 1 0 0 1 7

Cancer Genetics and Cytogenetics4, 169-177 {1981} 0165-4608/81/0601690952.50

170

I.B. Larripa and S. B. de Salum

MATERIAL AND METHODS Murine fibrosarcoma CS1, CS2, and CS3 appeared after the subcutaneous implantation of empty plastic or glass cylinders in BALB/c mice, after a latency of 9 - 1 2 months, and were classified histologically as anaplastic sarcomas indistinguishable from the tumors induced by the cylinder filled with human neoplastic cells; although, in this case the tumor incidence was significantly increased [2]. With this experimental design, 15 tumors were studied: three out of five tumors induced by empty cylinders and one of ten induced by cylinders with neoplastic cells inside; all tumors had the same markers. These tumors were very fibrous and compact, so much so that it was practically impossible to apply techniques of direct cytogenetic study. For this reason, cultures were established with the following technique. In each case a small piece of the tumor was washed in culture medium, containing antibiotics, and cut up with a scalpel. The minced material was seeded in flasks containing culture medium (MEM) plus 20% calf serum, 100 U penicillin and 100 ~tg of streptomicin in a final concentration of 1.5×10 s cells/ml. Thus, we obtained cell lines CS1, CS2, and CS3 from three different mouse tumors. For cytogenetic analysis, after exposing the cells for 4 5 - 6 0 min to Colcemid (1 ~tg/ml), 0.25% trypsin (Difco) was added to the culture flasks and the cells removed by gentle agitation. Hypotonicity was achieved by exposure to 0.075 M KC1 solution for 15 rain. Fixation in acetic metanol (1:3) was followed by air-drying at room temperature. The spreads were stored for a week and then processed by G banding, according to Seabright's technique [3] and C banding according to that of Arrighi and Hsu [4]. A total of 30 metaphases from each cell line were analyzed and the chromosomes arranged according to the standard karyotype of the mouse [5]. RESULTS

The cells of three cell lines, CS1, CS2, and CS3, grew in monolayers after 10 days in culture and h a d fibroblastoid aspects similar to those of the original anaplastic fihrosarcomas. Observations of cells from passage 4 to passage 65 with the Giemsa technique showed that the cells of the three in vitro cell lines presented heteroploidy ranging from 3 0 - 86 for CS1, 4 4 - 92 for CS2, and 42 - 93 for CS3, with modal numbers of 59 and 66 for the first and second. The CS3 cell line had modal numbers of 44 and 89 (Table 1). The three cell lines w e r e identified by the G-banding technique. Passages 10, 40, and 55 were studied for CS2, CS3, and CS1, respectively. It was determined that the pairs of chromosomes most affected by the presence of supernumeraries were chromosomes #10, #16, #18, and #19 in the four tumors studied. These results were obtained by analyzing ten banded karyotypes from each tumor (Tables 2-5). Table 1

Coincident markers in four cell lines

Modal Cell line ' No. CS1 CS2. . . . CS3 P193

59 66 44 89 69

Heteroploidy

DM

MLA(%) MM(%)

30-86 44-92

+ ÷

91 50

35 14

42-93 50~130

+ +

66 98

22 15

171

Table 2

The representation of the 19 mouse autosomes i n ten cells of the CS1 cell line~ N u m b e r of c o p i e s

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

1

2

3

2

6 9 4 4 7 7 4 4 4 4 3 3 8 3 4 1 1

2 1 4 3 1 2

2 2 2 2

4 2

1 2

2 2

1

2 1 1

4 4 1 4 6 1 4 2 2 2

4

5

6

7

8

1 1

1 2

3 1

1 1 1 2 3 4 1 1

2 2 3 4

1 2 1

1

1

1 1

1

Average per cell

D i ffe re nc e found - expected

2.0 2.1 2.2 2.5 1.9 2.4 1.2 2.2 2.5 4.0 2.5 2.8 2.3 2.6 2.8 4.5 3.6 4.5 4.6

--0.8 --0.7 --0.6 --0.3 --0.9 --0.4 --1.6 --0.6 --0.3 +1.2 --0.3 0.0 --0.5 -0.2 0.0 +1,7 +0,8 +1,7 +1.8

2.8 aThe marker chromosomes have been distributed among the different normal chromosomal types.

Table 3

The representation of the 19 mouse autosomes in ten cells of the CS2 cell line a N u m b e r of c o p i e s

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

1

2

3

4

1

2 6 9 6 5 7 5 8 3 1 5 7 4 3 3

4 2 1 1

2 2

3 3 1

1

1 1

2 1

5

6

7

8

1

2 2 5 2 6 2 2 2 4 5 3 2 3

1

1

2 3

2

2

1 2 2 2 4

1 3 1

1

2

4

1

3

1

1

3 1

2

2

Average per cell

Difference found-expected

3.1 2.6 2.1 1.8 2.1 2.1 2.5 2.2 2.5 4.8 2.8 2.1 2.5 2.9 3.4 5.0 3.4 4.4 6.1

+0.03 --0.47 --0.97 --1.27 --0.97 --0.97 --0.57 --0.87 --0.57 +1.73 -0.27 --0.97 --0.57 --0.17 + 0.33 +1.93 +0.33 +1.33 +3.03

3.07 aThe marker chromosomes have been distributed among the different normal chromosomal types.

172 Table 4

T h e r e p r e s e n t a t i o n o f t h e 19 m o u s e a u t o s o m e s i n t e n c e l l s o f t h e C S 3 c e l l l i n e a N u m b e r of copies

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

1

3 2 2

2 2 1

2

3

4

6 4 5 5 10 7 3 5 4 2 4 6 5 4 3

4 3 2 2

5

6

7

8

9

10

1 1

3

1 5 3 3 5 4 2 3 6 5 1 5 2

4

3

1

1

1

2

1

1

1 4 1 1 1

1 1 1 1

4 1

1 2 1

1 1

2 1

3

1

1

Average per cell

Difference f o u n d - expected

2.4 2.0 2.2 2.2 2.0 1.9 3.0 2.1 2.9 4.5 2.9 2.7 2.8 2.6 3.2 4.9 3.0 4.5 5.5

--0.62 --1.02 --0.82 --0.82 --1.02 --1.12 --0.02 --0.92 --0.12 +1.48 --0.12 --0.32 --0.22 --0.42 +0.18 +1.88 --0.02 +1.48 +2.48

3.02 ~The marker chromosomes have been distributed among the different normal chromosomal types.

Table 5

T h e r e p r e s e n t a t i o n o f t h e 19 m o u s e a u t o s o m e s i n t e n c e l l s o f t h e P 1 9 3 c e l l l i n e a N u m b e r of copies

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

1

2

3

4

5

1

4 3 1 5 5 6 4 5 5 1 4 5 4 3 2

3 4 7 2 3 3 4 4 3 3 3 4 4 4 3 1 6 3 1

1 2 2 1 2

1 1

1

1

2 2 2

1 1 2 3 3 1 2 2 2 3 1 1 1

6

7

8

9

2

1

2

1

1 1 4 1

1 1

1

3 2

1

2

1 1

Average per cell

Difference f o u n d - expected

2.7 3.1 3.1 3.0 2.7 2.2 3.0 2.6 2.7 3.9 2.9 2.8 2.8 3.1 3.2 4.8 3.1 4.4 4.8

--0.49 --0.09 --0.09 --0.19 --0.49 --0.99 --0.19 --0.59 --0.49 +0.71 --0.29 --0.59 --0.39 --0.09 +0.01 +1.61 --0.09 +1.21 +1.61

3.19 aThe marker chromosomes have been distributed among the different normal chromosomal types.

Coincidence of Cytogenetic Markers

173

All identified chromosomes were considered in the analysis, including those partaking in marker formation. Unidentified chromosomes were excluded. Sex pairs were also excluded, because of their heteromorphism. The number of copies of each autosome for the ten cells gave the observed incidence of each chromosome type. The average of these figures is the expected incidence value. Differences between found and expected indicate which chromosomes type gained or lost copies. The following markers were identified by G banding in the CS1, CS2, and CS3 cell lines: Double minutes {DM}: a chromosomal aberration also called minute chromatin bodies (MCB), microchromosomes, or minute chromosomes and consisting of an extremely small structure which appears in variable number in almost all metaphases (Fig. 1). When C banding was performed, the dm seemed to decrease and did not take the characteristic staining of centromeric areas (Fig. 2). Long acrocentric marker (MLA): MLA appeared in 91, 50, and 66% of the metaphases for the CS1, CS2, and CS3 cell line, respectively (Table 1). It can be considered as a translocation in tandem of chromosomes #1 and #16, t(lq;16q) {Fig. 3). Metacentric marker {MM): centric fusion between two #10 chromosomes, Rb{10.10), appearing in 35, 14, and 22% of the metaphases for CS1, CS2, and CS3 cell lines, respectively {Table 1} (Fig. 4). Submetacentric marker 1 (MSM1): centric fusion between chromosomes #1 and #18, Rb(1.18). Submetacentric marker 2 (MSM2): centric fusion between chromosomes #10 and #18, Rb(10.1S). Submetacentric marker 3 (MSM3): centric fusion between chromosomes #15 and #18, Rb(15.18). The latter three markers (MSM1, MSM2, MSM3) appeared sporadically in the cell lines. DISCUSSION Both dm and MLA have been found in four cell lines derived from foreign body sarcomas: three of them (CS1, CS2 and CS3) originated around empty cylinders and the other, P193, from a tumor induced by a cylinder into which human neoplastic material had been inoculated. The results of C banding in all lines (CS1, CS2, CS3, and P193) suggest that DM are acentric because of the lack of centromeric heterochromatin. With C and G banding the quantity of observed dm is reduced and the remaining ones stain as normal euchromatin. Levan et aL in lg76 [6] proved in mouse tumor cells that DM are not C heterochromatin. These authors suggested, judging from Cband analysis, that DM do ~ o t originate from centromeric regions of chromosomes. Barker and Hsu in 1978 [7] using the Cd-band technique in a cell line of human breast tumor, observed lack of one stained spot in the dm. Its permanence during long periods in vitro would a priori indicate that the dm are centric. Levan et al. in 1978 [8] demonstrated in SEWA ascites tumors of the mouse that most of the din, in spite of the lack of detectable centromeric activity, were included in telophase nuclei because they were enclosed by the nucleolar matter persisting around the chromosome ends. With the C banding the marker MLA and MM contained only the centromeric band. The HSR (homogeneously staining regions) and dm have been described in tumors, and both present similarities in G banding and they are negatively C hetero-

174

Figure 1

Metaphases showing DM chromosome aberration.

Figure 2 C-banded metaphase in which DM did not take the characteristic staining of centromeric areas.

q

q

;" ~ "

I'

-,'

i

;'.,i~

:,.'

J

176

I . B . Larripa a n d S. B. de S a l u m chromatic a n d not late-replicating [9]. We observed no HSR in our material, in spite of the fact that other authors m e n t i o n HSR as associated w i t h the presence of d m [10]. The HSR a n d d m are a p p a r e n t l y associated w i t h essentially the same mechan i s m s for the a m p l i f i c a t i o n of genes stimulating t u m o r viability [11,12]. Recent b i o c h e m i c a l e v i d e n c e has d e m o n s t r a t e d that parts of the g e n o m e in m u r i n e t u m o r cells a n d CHO cell lines resistant to methotrexate m a y undergo gene amplification. This m a y p r o c e e d in two ways: Ca) stable amplification, w h e n the genes are localizated to e x p a n d e d c h r o m o s o m e regions called HSR [13], and (b) unstable amplification, w h e n the genes are associated w i t h d o u b l e m i n u t e c h r o m o s o m e s [14]. Buoen a n d Brand [15] f o u n d DM in 5 of 70 m u r i n e t u m o r s i n d u c e d by plastic film ( p o l y m e r tumorigenesis). W e f o u n d DM in four of 15 tumors studied. The finding of DM is an interesting feature, w h i c h was first observed in the cells of a pleural exudate from a h u m a n p u l m o n a r y c a r c i n o m a [16]. At present, DM have been found in h u m a n tumors, m a i n l y of neurogenic origin, b u t being also detected in leukemia [17], colon cancer [18], ovarian c a r c i n o m a [19], a n d breast a d e n o c a r c i n o m a s [20]. In animals, DM was first seen by Mark [21] in Rous sarcomas of viral etiology. To date, d m have been f o u n d in about 30 e x p e r i m e n t a l tumors (mostly sarcomas), i n c l u d i n g four of this paper. The origin of DM is not w e l l u n d e r s t o o d [17], though it has been attributed to viral infection [22]. The fact that all four cell lines c o n t a i n e d the same markers suggests that they m a y be related to foreign-body tumorigenesis or viral activation a c c o m p a n y i n g t u m o r dev e l o p m e n t , or both, in mice bearing e n d o g e n o u s viruses [23]. N o n r a n d o m anomalies, both structural (DM, MLA, MM) a n d n u m e r i c a l (gains in c h r o m o s o m e s #10, #16, #18, a n d #19), f o u n d in the s t u d i e d m o u s e tumors are another e x a m p l e of the dependence of the c h r o m o s o m e pattern on the i n d u c i n g agent.

This work was supported by grants from CONICET (Consejo National de Investigaciones Cientificas y T~cnicas). We also acknowledge the valuable technical assistance of Mrs. E. de Licen and the photographic work of Mr. O. Miskoski. REFERENCES 1. Salum SB, Larripa I (1975): Minute chromatin bodies in a murine in vitro cell line. J Natl Cancer Inst 55, 717-720. 2. Pasqualini CD, Sen L, Saal F, Schwartz L, Tkaczevski LZ (1973): Tumor development in mice bearing a plastic cylinder and inoculated with human neoplastic cells. J Natl Cancer inst 51,263-286. 3. Seabright M (1971): A rapid banding technique for human chromosome. Lancet 2, 971-972. 4. Arrighi FE, Hsu TC (1971): Localization of heterochromatin in human chromosomes. Cytogenetics 10, 81-66. 5. Committee on standardized genetic nomenclature for mice (1972): Standard karyotype of the mouse, Mus musculus. J Hered 63, 69-72. 6. Levan G, Mandahl N, Bregula U, Klein G and Levan A (1976): Double minute chromosomes are not centromeric regions of the host chromosomes. Hereditas 83, 83-90. 7. Barker PE, Hsu TC (1978): Are double minutes chromosomes? Exp Cell Res 113,457- 458. 8. Levan A, Levan G (1978): Have double minutes functioning centromeres? Hereditas 88, 81-92. 9. Levan G, Mandahl N, Bengtsson BO, Levan A (1977): Experimental elimination and recovery of double minute chromosomes in malignant cell populations. Hereditas 86, 75 - 90. 10. Balaban-Malenbaum G, Gilbert F (1977): Double minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell llne. Science 198, 739-741.

C o i n c i d e n c e of C y t o g e n e t i c M a r k e r s

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