Hereditas 92: 259-265 (1980)

Large double minutes with ring-shape and rod-shape ALBERT LEVAN and GORAN LEVAN

Institutes of Genetics, Universities of Lund and Gothenburg, Sweden

LEVAN, A. and LEVAN, G. 1980. Large double minutes with ring-shape and rod-shape. - Herediras 92: 259-265. Lund. Sweden. ISSN 0018-0661. Received January 31, 1980

High incidence of large double minutes (DM) was characteristic of one subline of the SEWA mouse ascites tumor. This subline, SEWAIR, was the one in which the new C-bandless chromosomes (CM) appeared. The incidence of large DM was associated with low incidence of DM per cell, low incidence of DM-carrying cells, and high incidence of CMcanying cells. The large DM were either round or elongated (rod-shaped). Many of the former kind were suggestive of ring-shape and a few of the largest obviously constituted pairs of rings, free or interlocked. One configuration of 4 interlocked rings was evidently in its second mitotic generation; in the same cell another confguration of 2 pairs of interlocked rings had developed from 1 pair of free rings in the preceding generation. Possible relations between CM and large DM are considered.

Albert Levan, Institute of Genetics. University of Lund. S-223 62 Lund. Sweden

Nondisjunction during mitosis is a main cause of chromosome number variation in tumors. In typi- cal nondisjunction, failure of the centromeric function causes the two chromatids of a chromo- some to remain together during anaphase. At telo- phase they are both included in one of the daugh- ter nuclei or in a micronucleus. Double minutes (DM), which increasingly often are detected in malignant materials, have no centromeres and regularly undergo nondisjunction at mitosis. This is achieved by a peculiar mechanism by which both daughter halves of a DM become stuck in the nucleolar matter that envelopes the anaphase chromosomes and are thus transported into one of the daughter nuclei (BARKER and Hsu 1978; LEVAN and LEVAN 1978). The efficiency of this mechanism must be dependent on the size of the DM, and it seems reasonable to assume that selective forces are acting towards a DM size, optimal under the conditions prevailing.

Even though the DM of a tumor generally are of the same size, differences occur among cells. Also, several sizes may be found among the DM of the same cell, and the impression is that this va- riation in size is discontinuous and that different size classes may be discerned. Nothing is known about the mechanism underlying the size varia- tion. A priori it has been suggested (e.g. by LEVAN et al. 1978, p. 12) that the DM may have ring-shape with the potential to changes in size inherent in

this shape. During our work with the SEWA mouse tumor, we have occasionally seen unusu- ally large DM that have exhibited signs of being ring-shaped, but only recently have we come across a subline of this tumor with DM large enough to display indisputable ring-shape.

Material and methods The subline with many large DM, SEWAlR, was started in serial (intraperitoneal) in vivo transfer in April 1976 from cells of the in vitro line SEWAI, which had been set up in culture in December 1975 from a SEWA stock tumor (LEVAN et al. 1977, p. 77). SEWAlR originally contained some 90 % of DM-carrying cells, mostly with high numbers of small DM in each cell. During one specific pe- riod-between December 1976 and March 1977-SEWAIR underwent a dramatic change: The DM were substituted by a varying number of copies of a new chromosome type called CM (C-bandless, LEVAN et al. 1978). After this change most cells were without DM, but exceptional sin- gle cells with DM could be found. In these cells the DM were generally fewer and larger than usu- al. Samples of SEWAlR were stored in the cell bank in March 1977 directly after the discovery of the CM. Later on, the lines SEWAlR Recl and

260 A L E V A N A N D G LEVAN Hereditas 92 (1980)

SEWAIR Rec4 (called Recl and Rec4 below) were started in serial in vivo transfer from such samples, Recl in April 1977 and Rec4 in June 1978. As a "normal" control, a SEWA stock car- ried in strain A hosts has been used. This line was started in July 1976 and is still grown serially. It was selected for this purpose because its content of many small DM in nearly 100 % of the cells has kept constant all this time. It would have been preferable to use a SEWA stock in its syngeneic host, A.SW, but in our laboratory this line has exhibited fluctuations between periods with high incidence of visible DM and periods with few or none.

The material for the present study consisted exclusively of ascites cells grown intraperitoneally in vivo. Samples of the ascitic fluid were drawn from the hosts on the third day after inoculation of lo7 cells per animal and were directly processed for chromosome analysis as described in LEVAN et al. (1978). The chromosomes were usually stained by Giemsa. In our analysis we have counted the DM of each cell and divided them into 3 size classes: (1) small DM, which included barely vis- ible ones, sometimes perceivable only as a fuzzy background; (2) medium-sized DM, distinct, ap- parently round structures with diameter below the normal chromatid breadth; and (3) large DM, which were of 2 types: (a) round DM, and (b) elongated, rod-shaped DM. The largest of the former kind displayed clear ring-shape.

Incidence of large DM The incidence of CM, DM and large DM in the original SEWAIR, Recl and Rec4 is shown in Fig. la-c, in which the percentage fraction of large DM among the DM-carrying cells is marked by round filled dots against a background of the total per- centages of cells with DM (whole-drawn lines) and CM (broken lines). In Fig. la and b, the latter 2 curves show reverse relations between DM and CM: when one is low, the other is high and vice versa. Thus, the 2 curves cross each other: in Fig. la, DM went rapidly down after day 100, and CM went up. In Fig. Ib, the reverse events took place: at the start CM was high, DM low; after 100 days cells with DM rapidly replaced the cells with CM. These conditions illustrate the rule usually valid in SEWAlR derivatives: DM and CM behave as if they were mutually exclusive. This interesting point will be dealt with more fully elsewhere.

In Rec4 the situation was more stable; DM re- mained low and CM high during a period of 500 days (Fig. Ic). Rec4 is still carried in serial in vivo transfer, and the relation DM:CM seems to be well established. It is doubtful whether eventually DM will be substituted for the CM, as happened with Recl and another related strain. So far, Rec4 is the only one, in which a high level of cells with CM has remained stable for a long period.

Looking at the relations of large DM to the other parameters of Fig. la-c, it appears that there is a reverse association between the total percentage of cells with DM and the fraction of large DM. This is the usual situation in SEWA stock tumors with high incidence of DM. SEWA stock in strain A is an instance of such a tumor, 10 samples of which have been recorded during a 950-day period (Fig. Id). The frequency of DM-carrying cells was above 95 % all the time and that of large DM below 5 %. During last summer it has been shown by C-banding that this line-like all other SEWA lines so far tested-will occasionally exhibit sin- gle cells with CM, but they are quite rare, 1 in 50 to 500 mitoses.

In the following, the findings in Rec4 (Fig. Ic) will be examined somewhat more closely. Since its recovery from the cell bank in July 1978, this line has been sampled every 2 to 3 weeks. The I 1 samples that gave successful fixations have been recorded in the figure and form the basis of the analysis. Altogether 1903 metaphases were ana- lyzed, 178 of which (9.4 %) had from l to 2100 DM (Table I ) . Of these, 82 (46.1 %) had from 1 to 14 large DM, and 96 (53.9 %) only medium-sized or small DM. Of the 82 cells with large DM, 62 (75.6 %) had only large DM, while 20(24.4 %)also had medium-sized and/or small DM in the same cell. Of the 82 cells with large DM, 49 (59.8 %) had round and 29 (35.4 %) elongated DM, while 4 (4.9 %) had both types. It is seen from Table 1 that large DM were more frequent in cells with low number of DM; more than 96 % of the cells with large DM were below the total DM number of 9.

As pointed out above, most SEWAlR lines are characterized by a reverse correlation between the presence of CM and DM, most DM being found in cells without CM or with few CM. Such was the case with SEWAIR and Recl during the periods with few CM and many DM (Fig. 1). In Rec4, however, the frequency of CM was constantly high and no such correlation was seen. Also the large DM were distributed rather evenly over the different classes both of CM numbers and of total chromosome numbers per cell, as seen in Table 2 ,

Herediras 92 (1980) LARGE DOUBLE MINUTES 26 I

SEWAlR Rec 1 Rec 4 %

0 100 200

0 ..0.o.q

1

0 100 200

8%. 0.0.. . . .o 6 '0. . . . . . . . . . . . . . . . .o.. . .O.O.*. 0'"

00 0

0

0 0

0 0 .

0 A

100 200 300 400 6 0 0

Days a b C

SEWA stock in strain A O/O

'""1 d - -

- <

0 1.0, , , , , , & , , , e o , , , , I , = , 0 . - , q 100 200 300 400 500 600 700 800 900 0

Days d Fig. I . a-d. Percentage of cells with DM (whole-drawn lines) and CM (broken lines); filled dots show the percentage fraction with large DM among the DMcarrying cells; a: SEWAIR; b: Recl; c: R e d ; d: SEWA stock in strain A. Abscissa: percentage of cells; ordinate: number of days in serial in vivo transfer; day 0 September 22, 1976(a), May 3, 1977 (b), July 13, 1978 (c), April 14, 1977 (d); day 0 in b and c corresponds to day 182 in a , when the sample was frozen from which Recl and Rec4 were started.

in which the percentage fractions of large DM are recorded in relation to these 2 parameters.

SEWA stock in strain A, in which many DM were found; most of them were small, but some larger ones may also be seen. All the other pictures of Fig. 2 and 3 are from Rec4. Fig. 2b is a rnetaphase with 12 large DM of the round type. They had roughly the same size and clearly showed a tend- ency to ring-shape. In the microscope they ex- hibited a lighter area in their center, suggestive of a hole. Many instances of the large round DM

Morphology of large DM Instances of metaphases with large DM are pre- sented in Fig. 2 and 3. Fig. 2a shows a cell from

262 A . LEVAN A N D G . LEVAN Herediras 92 (1980)

Table I . Incidence of DM and large DM in Rec4

Number of DM (large DM) Total DM per cell

1 2 3 4 5 6 7 8 9 10 I I I2 14 I5 25 30 60 100 Cells DM

Number of cells with DM 47 34 23 17 10 13 9 2 4 1 2 5 4 2 1 2 I 1 178 918 5.16 with large DM 1 6 2 2 1 4 1 2 6 6 2 I - - - 2 I - - - - - 82 276 3.37

Percent large DM in single classes 34 55 61 71 60 46 22 50 - - - 40 25 - - - - - in groups of classes 53.4 31.0 23.1 0

showed a similar tendency, but only 7 of the 53 cells with large round DM contained quite obvious rings; 4 of these metaphases are presented here as Fig. 2c, d and Fig. 3a, b. In Fig. 2c there are 2 pairs of free rings, and in Fig. 2d, in which 4 size classes of DM occurred, the largest class was rep- resented by a pair of interlocked rings. The DM of the smallest class in this cell were seen very clear- ly in the microscope, but were difficult to make visible in the photographs. The cell of Fig. 3a had 1 pair of free rings and 1 of interlocked rings. The cell of Fig. 3b had interesting and revealing confi- gurations of interlocked rings. The lower one in the figure contained 4 rings, all interlocked but with 2 pairs of rings more intimately interlocked. The drawing in the insert is a possible interpreta- tion of this complex structure. Apparently this configuration stems from an interlocked pair of rings in the preceding cell generation. The upper configuration in the figure consisted of 2 pairs of interlocked rings, apparently originating from 1 pair of free rings in the preceding generation. The last 2 pictures of Fig. 3c and d present instances of cells with large, rod-shaped DM.

Discussion In the present paper interest has been focussed on the variation in size of DM, and special attention has been paid to the large DM that were frequent

in the SEWAIR subline, especially in the Rec4 line. I t must be recalled, however, that this is a rather specific material, not necessarily typical of all DM-carrying tumors. Rec4 was started as a sample from the SEWAIR cell population at the time when the new CM chromosomes had just been detected (see material and methods section). The CM are centric chromosomes and behave as chromosomes in most respects. In some respects, however, they resemble DM, as in their rhythm of DNA replication, their lack of C- and G-banding, their tendency to being eliminated in cell culture etc. (LEVAN et al. 1978). Also, there is some indi- cation that the centromeres of the CM are weaker than those of the ordinary chromosomes; some- times the CM tend to appear in pairs at metaphase, a possible sign of previous nondisjunction.

All in all, it is unquestionable that DM and CM are somehow related. At the present state of knowledge, it is impossible to discern exactly how. Have the CM developed from the DM or vice versa? Have both structures originated by amplification of specific loci in the ordinary chromosomes? Is it evenconceivable that they are of extrinsic origin? The production of large DM seems to be especially closely related to the pres- ence of CM. From the fluctuations in frequency of the large DM as well as from their variable ap- pearance (rods, rings, dots) it seems probable that in the Rec4 line they are continually produced but have a rather short life span. One might hypoth- esize that large DM arise from the occasional

Table 2 . Fractions of large DM in relation to the numbers per cell of CM and of chromosomes

Numbers of CM per cell: 0 1 2 3 4 5 6 1-10 Average Percent fraction of large DM: 30.0 62.5 56.5 47.2 42.4 38.5 42.7 46.2 45.3

Chromosome numbers per cell: 42-44 45 46 47 48 49 50 51 52-54 Average Percent fraction of large DM: 55.5 57.1 56.5 53.0 39.0 42.9 29.4 50.0 41.7 47.3

Herediras 92 (1980) LARGE DOUBLE MINUTES 263

Fig. 2 a-d. a: SEWA stock in strain A, passage 159, August 3 I , 1979, M: Rec4. passage 49, June 6, 1979 (b, c), passage 39. March 31, 1979 (d); a: many small DM, also a few larger ones; b: large round DM; c: large DM, 2 pairs of free rings; d: 12 DM of 4 size classes, including I large pair of interlocked rings, 8 large DM of 2 size levels, 3 small DM, hardly visible (arrows), the chromatids of one of them widely separated; inserts in c and d: larger magnifications of ring-shaped DM.

breakdown of CM, independently of the small The work of BALABAN-MALENBAUM and GILBERT (“typical”) DM. Statistical analysis, however, has (1977) in human neuroblastoma cell lines has dem- shown that cells containing both large and small onstrated that homogeneously staining regions DM are overrepresented or’ = 46.83; df = 1; (HSR) and DM may be related. In one cell line, the P< <o. 00 1 ) . cells exhibited either HSR or DM and the authors

264 A LEVAN A N D G. LEVAN Hereditas 92 (1980)

Fig. 3 a-d. Rec4, passage 49, June 6, 1979. a: I pair of free, 1 pair of interlocked rings; b: configuration of 4 interlocked rings, 2 pairs of interlocked rings; c: 3 large rod-like DM; d: 4 ditto; inserts in a and b: larger magnifications of ring-shaped DM, in b also a drawing with possible interpretation of the 4-ring configuration; arrows in c and d at large rod-shaped DM.

put forward the idea that DM arise through the breakdown of HSR. In our material, HSR are seen occasionally and we feel that CM may constitute a special case of HSR. Both HSR and DM have been functionally related to the production of in-

creased amounts of specific gene products (NUN. BERG et al. 1978; KAUFMAN et al. 1979).

Thus, interrelations between HSR, DM and CM appear to be well established in spite of our limited knowledge of the chromosomal mechanisms be-

Herediras 92 (1980) LARGE DOUBLE MINUTES 265

hind the transitions. We feel that these mecha- nisms must be previously unknown in eukaryotic cells, but possible parallels may exist in microbial systems. Transpositions of genetic material within and between bacterial chromosomes, plasmids and viruses exhibit at least superficial similarities to the DM system.

Acknowledgments.- Financial support of this work from the Swedish Cancer Society. from the John and Augusta Persson Foundation for Medical Research, and from the Cancer Interna- tional Research Co-Operative (CANCIRCO) is gratefully ac- knowledged.

Literature cited BALABAN-MALENBAUM, G . and GILBERT, F. 1977. Double

minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell line. - Science 198: 739-741

BARKER, P. E. and HSU, T. C. 1978. Are double minutes chromosomes? -Exp. Cell. Res. 113: 45f-458

KAUFMAN, R. J., BROWN, P. C. and SCHIMKE, R. T. 1979. Amplified dihydrofolate reductase genes in unstably metho- trexate-resistant cells are associated with double minute chromosomes. - Proc. Nat. Acad. Sci. 76: 5669-5673

LEVAN, A. and LEVAN, G. 1978. Have double minutesfunction- ing centromeres? - Hereditas 88: 81-92

LEVAN, A,, LEVAN. G . and MANDAHL. N. 1978. A new chromosome type replacing the double minutes in a mouse tumor. - Cytogener. Cell Genet. 20: 12-23

LEVAN, G . , MANDAHL, N., BENGTSSON, B. 0. and LEVAN, A. 1977. Experimental elimination and recovery of double minute chromosomes in malignant cell populations. - Heredi- tas 86: 75-90

NUNBERG, J. H., KAUFMAN, R. J., SCHIMKE. R. T., URLAUB. G. and CHASIN. L. A. 1978. Amplified dihydrofolate reduc- tase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. - Proc. Nor. Acad. Sci. 75: 5553-5556