Hweditus 86: 75---90 (1977)

Experimental elimination and recovery of double minute chromosomes in malignant cell populations GORAN LEVAN, NILS MANDAHL, BENGT OLLE BENGTSSON and ALBERT LEVAN Institute of Genetics, University qf Lund, Sweden

L~VAN G., . MANDAHL.N., BENGTSSON, B. 0. and LEVAN,A. 1977. Experimental elimination and recovery of double minute chromosomes in malignant cell populations. Hereditas 86: 75 -90. Lur.d. Sweden. ISSN 0018-0661. Received February 18, 1977 ~

The influence of the environment on the frequency of cells with double minute chromosomes (dms) was studied in the SEWA mouse ascites tumor. Under normal in vivo conditions, this tumor contained about 90”,, cells with one or more dms. Explantation in vitro resulted in a decrease of the dms carrying cells, which after 100 days only amounted to S”,, of the population. Following reimplantation in vivo. the original proportion of these cells was restored after an interval of 170 days. A mathematical model is presented describing these changes as selective phenomena. In the stock tumor almost all minute chromosomes were C-band negative. In the experimental populations. both in vivo and in vitro, another type of minute chromosomes appeared which were C-band positive. Also by other morphologic and functional criteria they were shown to be small chromosomes and not dms. In the present paper they have been referred to as microchromosomes. The fact that dms have been found only in malignant cells, and the correlation established in the present experiments between presence of dms and in vivo environment, led to speculations concerning their function. One possibility is that they involve amplification of genic material of significance to tumor development. Giirun Levan, institute qf Genetics, Universiiy of Lund S-22362 Lund. Sweden

It is interesting and suggestive that the strange chromosome-like structures called double minutes (dms) have been observed so far exclusively in malignant cells. Without claims of completeness, we have scanned the literature and found a total of 63 cases, in which dms have been reported, 38 of which were human and 25 animal malignancies. The fact that the dms have been restricted to tumors, and in certain cases have been maintained or even increased in frequency during serial in vivo transplantation (MARK1967, 1970), indicates that they play a role in the evolution of malignant stemlines towards ever greater efficiency in breaking down the defences of the host. This idea gains some support from 2 cases in the literature, in which dms, present in a high proportion of cells in early in vitro passages of the primary tumor, and thus also probably in the primary tumor, became completely or partially eliminated during prolonged in vitro growth. One of these cases was a Rous virus-induced mouse sarcoma (DONNER and BUBEN~K 1968), in which 92% of the cells of in vitro passages 2 and 4 contained dms in numbers from 1 to 60 per cell, whereas after 465 days in culture the dms had completely disappeared. “However, most of the mitoses showed 3-4 microchromosomes that had not been observed

in earlier examinations” (I.c., p. 87). The other case was a human neuroblastoma, established in continuous culture (BIEDLER et al. 1973). After 42 days in culture 70% of the cells contained dms, after 244 days this value was 14%,and “after approximately 1 year, cells with double-minute chromosomes were only occasionally found” (I.c., p. 2646). The latter case was inconsistent, however, in as far as 3 clones and subclones from the original culture contained high proportions of cells with dms (88-99%) after 149-205 days of culture. New cases ofcorrelation between in vivo conditions and presence of dms versus in vitro conditions and loss of dms were noticed during our work with rat sarcomas induced by benzpyrene (LEVANand LEVAN 1975 and unpubl.), in which we repeatedly observed that tumors with dms, when grown in vitro, tended to lose the dms. This was especially clear in one tumor, called BP2, in which 100%of the cells of the primaq tumor had from 1 to 14 dms. It was noticed that the dms varied in size from quite big ones, which were apparently centric, to extremely small ones, in which no centromeres were visible. With the experience we now have with the dms of the SEWA mouse ascites tumor we are convinced that the big minutes of BP2 were small ordinary chromosomes, while

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NOV 1

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Fig I The reldtionship between the different SEWA lines in vivo (dnimdl symbols). in suspension culture (standing bottles) and in monoldyer culture (lying bottle)

most of the small ones were true dms. This is the same difference as Donner and Bubenik noticed between their microchromosomes and the dms. For practical reasons we shall adopt their term “microchromosomes” for the minute chromosomes, which differ from the dms mainly in having a clear centromeric structure, holding the sister chromatids together at stages, when the chromatids of the dms are separated more or less widely. While dms have only been found in malignant materials, the microchromosomes are ubiquitous. In the BP2 the difference between microchrornosomes and dms became obvious during serial culture, when the former remained more or less stable in the populations, while the latter gradually became lost. When the experiment was interrupted at passage 45, the microchromosomes were only slightly fewer than in the primary tumor, 0.84 per cell versus 1.20, whereas the dms decreased by a factor of 10 during the same time, from 3.34 per cell to 0.32. Similar observations were made in another rat sarcoma, BP21, not included in the paper by LEVANand LEVAN(1975). In this tumor the microchromosomes were found in an average number of 1.78 per cell in passages 1-10 in vitro, and 1.80 in passage 65, the

same values for the dms being 2.67 and 0.50, respectively. It seemed highly tempting to test by systematic experiments whether these indications of a correlation between malignant in vivo growth and presence of dms were accidental or expressed a general trend. Having recently come across an excellent dms carrier, the SEWA tumor of the mouse, we planned some experiments to study the dms in different environments. In the following, experiments will be reported, dealing with the dms in the ascites fluid in vivo, after explantation in vitro, during serial passage in vitro, and during serial passage in vivo after reimplantation of in vitro-grown cells into the animal.

Material and methods The history of the SEWA tumor and the chromosome methods were described by LEVANet al. (1976). The SEWA cells were easily adapted to suspension culture. The cells started growing immediately after transfer to Eagle’s Basal Medium with Hanks’ Salts supplemented with 15% Fetal Bovine Serum.

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Population doubling time was determined in December 1975 to 4.6 days. It was possible, by selecting cells which remained attached to the surface of the plastic bottle, to obtain a cell type which preferred growing as a monolayer. The following SEWA materials were used in the present study: ( I ) SE WA stock tumor. We received passage 48 of this tumor from Professor George Klein's laboratory in December 1975 and have continued carrying it by weekly transplantations in the abdominal cavity of A S W mice. (2) SEWAI. -This is a suspension culture, started from the stock tumor on December 18, 1975. This line is still maintained in culture and was studied continuously until April 15, 1976, and after that occasionally. It has been retransplanted into animals several times and has immediately started growing as an ascites. Thus, on April 4 it started an ascites tumor called SEWAlR, which is still carried in serial in vivo passage. On October 27 and November 11, 1976, 2 more ascites lines called SEWAIR2 and SEWAIR3 were started from passages 48 and 50 of SEWAI. (3) SEWA4 und SEWA7. - Several more suspension cultures have been started at different times. Two of them, SEWA4, set up on February 12, and SEWA7, on March 19, 1976, were occasionally sampled for chromosome purposes. (4) SEWAmono. When SEWAI was passed for the first time, on December 22, 1975, a monolayer culture was isolated, called SEWAmono. It was implanted into animals on April 22, 1976, and is still carried as an ascites under the designation SEWAmonoR. The relationships between the different SEWA lines are represented in the diagram of Fig. 1.

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Observations 1. Chromosomenumbers

The SEWA tumor is hyperdiploid with the stemline number 43. In Table 1 the chromosome number distributions of the stemline region are given for the stock tumor (from LEVANet al. 1976, Table 1, p. 86) and for the experimental materials of the present study. As just described in the preceding section, these latter included 4 strains in serial culture in vitro, 2 ofwhich -SEWAI and SEWAmono -were also followed after retransfer into serial passage in vivo, now under the names SEWAlR and SEWAmonoR. Usually the values of each strain were based

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Chromosome number

Fig. 2. Chromosome numbers, averages of entire material; whole-drawn line: SEWA stock; dotted line: SEWAI. 4, 7 and mono in tissue culture; broken line: SEWAI R and monoR after reimplantation into animals.

on several samplings, as listed in the third column of the table. Most samples had 43 as stemline number, the only exception was the second passage of SEWAI R, which had unusually high chromosome numbers (stemline number 45, mean number 44.9). Also the later passages of this material had chromosome numbers above average (means: 43.4-43.8). Thus it is possible that in this case the change from in vitro to in vivo conditions was associated with a slight increase in chromosome number. No such change was seen in SEWAmonoR as compared with SEWAmono. One feature common to all the experimental materials as compared with the stock tumor, was their much less pronounced modes. While the modal number of the stock tumor contained 54% of the cells, the modes of all the other materials had values between 22 and 34%, and their modal regions were accordingly flatter and broader. This should be a sign of less stabilized conditions in the experimental materials. The 3 average distributions of SEWA

stock, of the combined in vitro materials (SEWAI, 4, 7 and mono) and of 2 materials carried back in vivo (SEWAIR and monoR) are presented in Fig. 2. Other signs indicating lowered chromosome stability were the frequent occurrence of polyploid cells, as also of various mitotic disturbances including chromosome breakage. Polyploidy, which was quite low in the stock tumor and seldom reached So/>, varied between 6 and 36% in 11 samples of various of the in vitro materials. In some samples micronuclei were frequent and, in association with them, pulverization of single chromosomes, also signs of mitotic disharmony. 2. The behavior of the dms

The most complete experiment of changed environment was carried out with SEWAl and SEWAIR. The proportion of cells with various numbers of dms in this experiment is presented in the diagram of Fig. 3. In this diagram each bar represents one

f f i w t l i l u s86 (1977)

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SEWA

Daystrornstart: PassageNo.:

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27 34

8 9 14 15 18 20 47 40

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128 141 158 167 186 249 278 291 362

Fig. 3. Percentage of cells with different numbers of dms, from above: no dms (white), 1-10 dms per cell (light-gray), 10 50 (darker gray), more than 50 (black). The line above the bars indicates mode of growth: ascites tumor in vivo (solid line), in vitro culture (dotted line). From left to right: SEWA stock (bar I), SEWAI. explanted in vitro on December 16, 1975 (bars 2-1 I ) , SEWAIR reimplanted in vivo (bars 12-21). Each bar is based on at least SO cells (except b a r 2 with 36 cells).

sample. From left to right these samples are: the SEWA stock tumor, 10 samples of SEWAl covering passages 2 to 47 in suspension culture, 10 samples of SEWAl R after reimplantation in vivo, covering passages 0 to 22 of the ascites tumor. At the construction of each bar of the diagram, the underlying sample was divided into 4 classes of cells, having the following numbers of dms per cell (from above): (1) None, (2) 1-10, (3) 11-50, (4) more than 50. The diagram speaks for itself: In the stock tumor, some 20% of the cells had more than 50 dms and some 10% were without visible dms, the rest had intermediate numbers. As soon as the cells were transferred into tissue culture, the zero class started growing, at first slowly (passages 5-8), then more rapidly (passages 9-15), and at passage 18 comprised nearly the entire population. During the period we took regular samples of SEWAl for chromosome analysis, that is up to passage 20, dms carrying cells were never completely lost; if large enough samples were scanned, such cells would always be found. Subsequently, on October 20 (day 307 after explantation in vitro), passage 47 of SEWAl was studied. At that time no true dms were seen in 185 cells examined. As described in the material and methods section, passages 48 and 50 of this culture

were implanted in vivo on October 27 and November 11, respectively, thus on days 314 and 329 after explantation, resulting in the ascites lines SEWAl R2 and R3. They were both sampled for chromosomes on November 30, after 24 and 19 days in vivo, respectively. In each of them 1 cell was found with dms: in SEWAIR2, 1 cell with 25 dms among 83 cells examined, in SEWAlR3, 1 cell with 12 dms among 107 cells examined. This indicates that even passages 47-50 of SEWAl might not be completely devoid of cells with dms. The second phase of the experiment, reimplantation of the practically dms-free cells into the body cavity of mice, started on April 4. Samples of 5 x lo6 cells of SEWAl passage 18 were inoculated into 2 A S W animals. Five days later, ascites started coming up and on April 26 the tumor was passed. Passages 1, 2, 3 and 5 in vivo were fixed and found to have the same low proportion of cells with dms as the parental in vitro sample. The proportion of cells with dms was somewhat higher in passage 7, and in passage 12 in late August (141 days after implantation in vivo) some 30% of the cells carried dms. At the time of the next sample, 1 month later, conditions approached the original in vivo situation, and the 2 last samples examined, passages 18 and 22

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Fig. 4. Percentage cells with one o r more dms under in vivo conditions (filled-in symbols) and in vitro conditions (open symbols); circles: SEWA stock-SEWAl-SEWAl R (circles belonging to the main experiment joined by lines); squares: SEWA4; triangles: SEWA7; hexagons: SEWAmono-SEWAmonoR; upturned arrow: change from in vivo t o in vitro; downturned arrow: change from in vitro to in vivo.

(183 and 254 days after the implantation in vivo) had a proportion of cells with dms as high as in the stock tumor. Another diagrammatic representation of the same experiment is given in Fig. 4, in which the development of the cell fraction with at least 1 dm has been plotted against time (number of days from the start of the experiment). This curve suggests a lag period immediately after each change of environment, lasting some 30 days after transfer into in vitro conditions and some 50 days after transfer back to in vivo conditions. After the first lag period it took some 70 days in vitro to get rid of most cells with dms and after the second some 130 days in vivo to get them back again. The other experiments were more fragmentary but still important. All observations in them were in agreement with the main results of the SEWAISEWAl R experiment. Thanks to this corroboration, the conclusions carry more weight. The additional samples analyzed have been represented in Fig. 5, which is arranged in the same way as Fig. 3. These samples include 5 in vitro samples of the 2 suspension culture strains SEWA4 and SEWA7, and 2 in vitro samples and 1 sample after reimplantation in vivo of

SEWAmono. The last bar of Fig. 5 is a sample from SEWA stock grown in a foreign strain of mice. The samples of SEWA4 and SEWAI exhibited the same general trend as just described in SEWAI. When plotted against time in Fig. 4, they coincided fairly well with the SEWAI curve. The third group of bars, SEWAmono (2 bars) and SEWAmonoR (1 bar) belonged to the strain selected for capacity of the cells to attach to the glass. The 2 in vitro samples, from days 28 and 92 after explantation, differed from SEWAI after the same period in vitro in having higher proportions of cells without dms. When plotted on the coordinate system of Fig. 4, it became evident that both samples, and especially the first one, had lost the dms much earlier than the suspension culture of SEWAI. The sample of SEWAmonoR, studied after 13 passages and 164 days in vivo, contained a very high proportion of cells with dms: in 145 cells only 6 were without dms. In comparison with the SEWA stock (Fig. 3, bar to extreme left) the proportions of the 4 classes apparently had changed. The SEWAmonoR sample also deviated from the SEWAI R samples from the same time (Fig. 3, bars to extreme right). The most obvious difference was the deficit in SEWAmonoR

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Fig. 5. Percentage of cells with different numbers of dms; same arrangement as in Fig. 3 From left t o right: 2 different SEWA explantations in vitro (bars 1--5). SEWAmono (bars 6-7), SEWAmonoR (bar 8) and SEWA stock in vivo in A strain mice (bar 9).

of the extreme classes (more than 50 and zero). To elucidate the difference a little further, the exact numbers of dms were determined in 100 cells of SEWAmonoR and the distribution resulting compared with that of the SEWA stock (counted in LEVANet al. 1976, Fig. 1, p. 85). The result is summarized in the diagram of Fig. 6, in which the following consistent differences between the 2 materials were noticed: SEWAmonoR had less cells than SEWA stock without dms and with more then 40 dms, and more cells with 1 to 40 dms. Even though, naturally, no definite conclusions can be drawn from just one sample, the difference was striking and it should be observed that SEWAmonoR also differed from the suspension culture in another point, the rate of loss of dms after explantation in vitro. Evidently, variations in the in vitro environment of the SEWA cells may effect adjustments of the composition of the population. That variations in the frequency of dms can also be effected by changes in the in vivo environment is shown in the last bar of Fig. 5, which represents ordinary SEWA stock cells, grown for 5 months or 21 transplant generations in the foreign strain A mice. This experiment will be reported in full in a later paper; at present this sample is included to illustrate that the host genotype is capable of influ-

encing the proportion of cells with dms. In the sample of Fig. 5, none of the 100 cells analyzed was without dms, and very few cells had only single dms. More than half of the cells had above 50 dms, many of them several hundred. In summary of this section, the present experiments have demonstrated that the incidence of dms in the SEWA population is dependent on whether the cells grow in vivo or in vitro. The high incidence of dms, which characterizes the ascites tumor in vivo, and which has apparently persisted since 1973, when dms were first observed in this material by FENYO et al., fades away in cell culture in vitro. This was shown in 4 experiments. After transfer back into in vivo conditions, the dms gradually return. This was shown in 2 experiments. The incidental observation that the frequency of dms was remarkably high after growth in a foreign mouse strain i s interesting and will be tested further. 3. Dms and microchromosomes It was pointed out in the introduction that in addition to the dms there exist chromosome structures of the same size order but different from the dms in many points. We decided to refer to them as microchromosomes.

G . LEVAN ET AL.

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No. of DMS per cell Fig, 6. Percentage of cells (ordinate) with different numbers of drns (abscissa) in SEWA stock (filled circles) and SEWAmonoR (open circles).

The microchromosomes are usually larger than the dms, but in SEWA some dms are of the same size as microchromosomes o r even larger. The microchromosomes show clear signs of being centric, whereas even large dms do not. In the microchromosomes the anaphase separation of the chromatids is synchronized with the mitotic rythm of the ordinary chromosomes, while the chromatids of the dms separate prematurely. In the SEWA tumor, and probably in all tumors in species with centromeric heterochromatin, safe distinction is possible between microchromosomes and dms, the former being Cband positive, the latter not. Furthermore, in contrast to the dms, the microchromosomes are not lost during in vitro culture; there is even some evidence that they tend to increase in number. DONNER and BUBEN~K (1968) thus found 3 microchromosomes and no dms after a long period in culture of a tumor that during early in vitro passages had plenty of dms but no microchromosomes. In our SEWA materials the same trend was noticed repeatedly. In the stock

tumor microchromosomes were rare: the scanning of 2 C-banded slides revealed only 1 or 2 C-band positive microchromosomes (LEVAN et al. 1976, p. 89). In several of the cultures, however, microchromosomes were frequent, which need not mean that microchromosomes were specifically favored by the in vitro conditions. It may just be a sign that the populations were under stress in consequence to the change of environment. It may be a response similar to the widening of the stemline mode, the increase of the fraction of double stemline cells, the occasional shift upwards of the chromosome number etc. Table 2 gives instances of the frequency of cells with microchromosomes in some of the SEWA lines. The appearance of microchromosomes as compared with dms is shown in Fig. 7 and 8. Fig. 7 presents instances of microchromosomes and dms together with the largest ordinary chromosome from 3 rat cells (a-c) and 3 mouse cells (d-0. The former were from a late in vitro passage of the benzpyreneinduced rat sarcoma BP2. Almost all cells of this

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7uhlr 2. Percentage distribution of cells with 0-3 microchromosomes in certain SEWA lines Line

Passage Number of No. michrochromosomes 0

SEWAl SEWAmono

47 6 15

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1

40 53 84 10 57 28 88 12

2

15 ~

from the same SEWAmono culture, one (c, d) from the near-diploid region, the other (e, f) from the neartetraploid. Neither of them had any dms, but the former had 2, the latter 5 microchromosomes. The big marker chromosome, observed by FENYO et al. (l973), with a secondary constriction, appearing as an interstitial C heterochromatin in C-banding, was seen in all 3 cells of Fig. 8 (arrowheads). The conclusion of the present section is that the genuine dms are something essentially different from genuine microchromosomes both structurally and functionally. In mouse tumors there is, thanks to C-banding, a safe method to distinguish between the two. An interesting functional difference between dms and microchromosomes is the tendency of the former to disappear in vitro and accumulate in vivo, while the latter are indifferent to these changes in environment or, if anything, tend to accumulate in vitro.

Number Mean of cells per cell

3

4 4

3 2

0.71 0.24 0.58 0.12

1RS 50 100 100

~~

passage contained 1 or 2 microchromosomes, but only few cells had 1 or seldom more dms. The microchromosomes often had a constriction in the middle, suggesting a centromere with median location. Fig. 7d-f were from 3 SEWA cells, d and e from SEWAmonoR in vivo, f from SEWAmono in vitro. The latter chromosomes were taken from the cell photographed in Fig. 8a and b. Among the small structures in this cell one of the larger was assumed, even when examined in plain Giemsa, to be a microchromosome, and this was verified after C-banding (Fig. 8b, arrow). The other 2 cells of Fig. 8 were

x

83

Discussion Taken together with some occasional earlier observations. the results of the present experiments have

0'

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a Fig. 7a-f. Longest chromosome, one microchromosome and instances of dms from rat sarcoma BP2, in vitro passage 45 (a-c). SEWAmonoR, passage 13 in vivo (d-e), and SEWAmono, passage 15 in vitro (0.

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established an interesting trend of the dms: they persist in malignant cells in vivo but become eliminated, when these cells are grown for some time in vitro. The present facts undoubtedly suggest that the dms exert a favorable influence on malignant growth. This can be concluded from their exclusive occurrence in connection with malignancy as well as from their return into the populations after reinoculation in vivo of cells that have been drained of their dms during previous growth in vitro. This suggests that some factor in the host directed against the tumor is weakened by the presence of dms. An observation pointing in the same direction is the unusually high incidence of dms in a SEWA line grown for a long period in a foreign host genotype, in which the resistance against the tumor cells should be especially high. On the other hand, SEWA cell populations even with very low content of dms-carrying cells, in some cases practically devoid of such cells, have proven to be fully malignant. This shows that presence of dms in the SEWA cells is no prerequisite for their oncogenicity. It can only be the question of an additional stimulation by the dms, giving dms-carrying cells a slight selective advantage. In this connection reference is made to the following chapter of this paper, in which it is demonstrated that the changes in proportion between cells with and without dms during the SEWAl-SEWAIR experiment (Fig. 4) are in good agreement with the hypothesis that cells with dms have a certain selective advantage under in vivo conditions and corresponding disadvantage under in vitro conditions. In most experimental cancers, the stemline starts out with normal karyotype. The tumor may grow for a long time without any chromosomal adjustments entering the stemline. Still, sooner o r later such changes will take place. In experimental tumors in mice and rats, these changes often consist of trisomy for one specific chromosome. In other cases, as in the best known human malignancy, the chronic myelogenous leukemia (CML), the initial change is not associated with any visible change in chromosomal quantity. It has been shown, however, that the progressional changes during the blastic crisis of CML involve very specific quantitative chromosomal changes, as the addition of a second Ph’ chromosome, a third No. 8. a third long arm of No. 17, thus quantitative changes that must stimulate

Hrrrdirm 86 (1977)

viability of the malignant cells. Another instance: several cases of human malignant lymphoma have been characterized by a medium-sized isochromosome marker, sometimes present in more than one copy, in one case even in 3 copies in all stemline cells. Here the 6-fold repetition of the same chromosome segment evidently stimulated tumor progression. In other tumors, as in human meningiomas, loss of specific chromosomes has been a characteristic feature. In general, quantitative changes within the normal karyotype are common in all kinds of malignant development. They should all have the effect of upsetting the original balance of the normal genotype, and if normal growth depends on the maintenance of normal genotypic balance, both loss and addition of chromosomes or chromosome segments may result in a step towards malignancy. Such systems have been worked out in detail and tested by large amounts of experimental data in mice and Syrian hamsters by Leo Sachs and collaborators (e.g. YAMAMOTO et al. 1973). During the last few years an interesting type of homogeneously staining chromosome regions (HSR) has been discovered in various malignant materials and also in one material of nonmalignant origin. This latter material was 13 antifolate-resistant Chinese hamster cell lines studied by BIEDLER et al. (1974). In 7 of these, in which the production of dihydrofolate reductase was increased more than 100-fold, long nonbanded chromosome segments were observed. At the same time LEVAN(1974) described and pictured such segments in one benzpyrene-induced rat sarcoma, in which certain markers had large grayish segments without bands and not resembling any part of the normal rat karyotype. LEVANand LEVAN(1975) reported further cases, and LEVAN ( I 975) summarized the observations as follows: “whereas most markers were formed from complete chromosome arms or large segments thereof, some marker chromosomes would contain considerable segments with G-band patterns that were incompatible with any normal chromosome segment of the same size. Often these segments would stain grayish in G-band staining and show no distinct bands. In one case, C-banding was attempted and it was found that in C-banding properties these segments did not differ from normal euchromatin” (I.c., p. 14). BIEDLERand SPENGLER(1976a. b) presented detailed descriptions and pictures of HSR both in

Fig. 8a-f. SEWAmono, passage 15 in vitro; a, b: cell with 42 chromosomes, 1 microchromosome (arrow). some 80 dms, 4 of which were larger; c, d: 39 chromosomes, 2 microchromosomes; e, f: 79 chromosomes, 5 microchromosomes: a, c, e: ordinary Giemsa; b, d, f: same cells in C-banding. Big marker chromosome with interstitial C-band at arrowheads in all 3 cells, 2 such markers in e-f.

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their antifolate-resistant Chinese hamster cell lines and in human neuroblastoma cell lines. Among 4 neuroblastoma lines studied, 2 had long HSRs in specific locations of the karyotype. Recently NIELSEN (1976) reported other cases of HSR in strains of the Ehrlich-Lettre ascites tumor of the mouse, carried in vivo since 1903. The fact that in her material the HSRs were located preferably in marker types that had shown high stability during the last 25-year period was taken as a sign that they acted favorably on the viability of the tumor. This was in accord with observations of LEVANand LEVAN(1975) that some HSRs had the same location in markers found in several different tumors. On the basis of their finding that a correlation existed between structure and function in the 7 HSR-carrying Chinese hamster cell lines, BIEDLER and SPENGIERspeculated that the origin of HSR “is the cytological consequence of some process of chromosomal amplification or magnification” (1976a, p. 187). Such a mechanism, by which specific genes are multiplied should be capable of changing the genotypic balance to meet specific challenges. These may include defense mechanisms of the host towards malignant growth. Obviously there are similarities between HSR and dm. In G-banding the dms stain homogeneously grayish without any bands even in large dms (G. LEVAN,unpubl.). The dms are negatively C-heterochromatic like most HSRs so far tested (LEVAN1975; BIEDLERand SPENGLER1976a, b; NIELSEN1976). It is true that the HSRs of the Chinese hamster cell lines stained as C-heterochromatin; they were, however not late-replicating, as neither were the HSRs of the neuroblastoma lines. It was demonstrated by autoradiography that both in the Chinese hamster and the neuroblastoma lines HSR replicated rapidly and synchronously and that in one Chinese hamster cell line replication of the HSR was completed before the midpoint of the S phase ( BIEDLERand SPENGIER 1976a, b). The dms so Far studied replicate at the same time as the euchromatin and early in the S phase (Luns et al. 1966; G. LEVAN unpubl.). The similarities in structure and behavior between HSR and dm make it tempting to regard them both as mechanisms to achieve the same result: amplification of gene sites changing the genotypic balance in favor of increased oncogenicity. Even though the present experiments are still in their beginning, they have undoubtedly elucidated certain new aspects of the confusing problem of the dms. The possibility they have indicated of eliminating the dms from cancer cells or accumulating them is promising. It is now possible to obtain at will dms-free as well as dms-enriched lines of SEWA and of other dms-

carrying tumors. This should facilitate the continued exploration of the dms.

Ma thematical treatment It is shown in Fig. 4 how the frequency of cells with at least one dm decreases in vitro and increases in vivo relative to the frequency of cells without dms. In the present chapter we shall investigate whether these results can be explained by the dms carrying cells having a lower growth rate in vitro than cells without dms, but a higher growth rate in vivo. However, a model based only on these assumptions can not give an adequate picture of the results obtained in the SEWAI-SEWAIR experiment. From this model it is expected that the frequency of dms carrying cells in vivo should increase until they dominate the whole population, just as the cells without dms do in vitro. This, however, is not the case; they increase only until they constitute about 90‘’:, of the population. A factor which can produce this kind of effect must, thus, be included in the model. It will therefore be assumed that dms carrying cells give rise, at a low and constant rate, to cells without dms. One possible biological interpretation of this feature of the model is: due to mitotic nondisjunction in cells containing dms, a certain fraction of daughter cells is produced which completely lacks dms. It will now be shown that a model based on these assumptions can produce evolutionary progressions which are similar to the ones obtained in the SEWAl--SEWAI R experiment. The values which must be given to the parameters of the model to produce these results are biologically plausible, and the different parameter values corresponding to the different experiments are consistent with each other.

I . Model

Assume that at time t the population of cells -which may be in vivo or in vitro - consists of N , cells without dms and M , cells with dms. Let their different growth rates be such that one day later, at time t + I , the cells without dms have given rise to k, N, cells and the dms carrying cells to k MM, cells. 3~ is defined as (kN/kM)-1. Thus, ci is positive if the cells without dms grow faster than the cells with dms and negative in the opposite case. Following the discussion above, we will assume that all descendants of dms free cells remain dms free.

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DOUBLE MINUTE CHROMOSOMES

and that a fraction, m, of the k M, cells has become dms free. The composition of the cell population at time. t t 1 can then be written N , + l= k N N , + m k M M ,

(]A)

M,+ I

(1B)

-m)kM Mi

To describe the evolutionary process, to which this simple model gives rise, the variable z,=N,/M, will be used. If z, increases with time, this means that the fraction of cells without dms in the population increases, and vice versa. Using the definition of tl made above we obtain

and a + m z,+---. m A z , = z , + -z,=--~ I-m I-m

(3)

87

2. SEWA1-SEWAlR

The question is now: Can values for C, CI and m be such that the function (4) describes evolutionary progressions similar to the ones occurring in the SEWAI in vitro experiment and the SEWAIR in vivo experiment? In the attempts to find such values, the experimental data were plotted in diagrams and then tested with different values on C, CL and m, until a good agreement was obtained between the data points and the curve given by equation (4). (In formal terms we have used the least square method to obtain the best fit between the curve and the data. The parameters have been determined with two significant figures.) For every time the cell population has been scored

An approximate solution of this difference equation is

Tuhle 3. The original and the transformed data for the SEWAI experiment

z, =C exp(t(cr + m)/( I - m)) - m/(cx+ m).

Day

(4)

When the three parameters C , CI and m are given definite values, the composition of the population, measured by the number of dms free cells divided by the number of dms carrying cells, is described by (4) as a function of time. C is a parameter of little biological interest; it describes when the evolutionary process begins. For example the experiment with SEWAI in vitro starts at day 0, while the in vitro experiment starts at day 108. By choosing correct values on C in the different cases, we can make the theoretical model “start” at the different days. c i and m have been introduced above. ry is a measure of the difference in growth rate between the two cell types and should not be much greater than lo?:, to have a biologically reasonable interpretation. m is the rate with which dms carrying cells give rise to cells without dms and should presumably be fairly small. It can be argued that m should change with the amount of dms in the dms carrying cells; cells with only few dms give presumably rise to cells without dms more often than cells with tens or hundreds of dms. For the sake of simplicity, this effect is not taken into account in the present model. We will discuss later what effect this simplification may have. (The model used here is presented as a difference model, since this is the simplest way to introduce the parameters. It is easy to change the model into a time continuous model which will give exactly the same results.)

Number of cells

z =N/M

y = In( 1002)

0.123 0.241 0.275 0.277 0.283 0.730 3.545 4.556 25.000 15.667

2.51 3.18 3.31 3.32 3.34 4.29 5.87 6.12 7.82 7.36

X

%

without dms with dms (N) (M) ~~

0 12 27 34 40 47 86 92 I06 I I9 307

43 7 11

26 26 54 39 41 50 47 I85

351 29 40 94 92 74 II 9 2 3 0

Tuhle 4 . The original and the transformed data for the SEWAl R experiment Day

Number of cells

z =N/M

x = In( 1 OOz- ’)

(0 without dms with dms (M) (N) 108

128 141 158 167 186 249 278 291 362

50 152 107 125 104 115 79 73 42 19

2 5 4 3 4 12 36 324 247 129

25.000 30.400 26.750 41.667 26.000 9.583 2.194 0.225 0.170 0.147

I .39 1.19 1.32 0.88 1.35 2.35 3.82 6.10 6.38 6.52

88

G . LEVAN ET AL.

I

0

b

25

50

75

100

125

t

Fig. 9. The SEWAI experiment; t =number of days, see Table 3 for the definition of y.

it is possible to compute the observed z value, by dividing the number of observed cells without dms with the number of cells with dms. We have, however, chosen not to plot the data in a diagram with z on one of the axis, but to transform the z values into what we call x and y values. The purpose of these transformations was to convert the data from both experiments -the SEWAI in vitro and the SEWAlR in vivo - into values which should fall on straight, increasing lines, if the dms carrying cells grew more slowly than the dms free cells in vitro, and more rapidly in vivo, and provided the two cell types did not convert into each other. It turned out in both cases that some of the initial observations must be ignored if a good agreement between the data and the curve was to be obtained. This omission is justifiable biologically; it took a few generations after the change in environment, before the cells started to act in a "normal" way. The original data and the transformed values for the SEWAI and the SEWAIR experiments are given in Tables 3 and 4. The y values obtained for the SEWAI experiment are plotted in Fig. 9, and the x values obtained for the SEWAIR experiment are plotted in Fig. 10. In the two figures are also included the two curves which give the best fit to the data points. The parameters for the curve drawn in the SEWAI case are C=0.065, a=0.047 and m=0.0017. These values are chosen to agree with the data from and including day 27. The observation at day 307 that no

dms carrying cell was found among 185 cells scored can not be plotted in the diagram since the point falls at infinity. According to the model the frequency of cells with dms should at this time be approximately one in 200,000; the agreement between the observed and the expected is thus good also for this day. The parameters for the curve drawn in the SEWAlR case are C=33,000, a=-O.O47 and m=0.0047. These values give the best possible fit between the curve and the data from and including day 158. 3. Discussion of mathematical treatment (1) There is no way to objectively decide whether

the fit between the curves and the data points is good or bad. Considering all the factors which may influence the observed values - from the quality of the fixations to the sampling effects at the scoring of the chromosome preparations or even at the passing of the cells - it is, actually, surprising that the data agree with the theoretical expectations as well as they do. This is particularly the case in the SEWAI experiment, in which eight (not counting the observation from day 307) values fall very close to the straight line which the curve approximates for the relevant values on t and the parameters. Even though the fit is a bit less good in the SEWAI R experiment, it is obvious that the model can fit in a very nice way both the phase of rapid increase of dms carrying

Hcwdrrus X6 (IY77)

DOUBLE MINUTE CHROMOSOMES

89

Fig. 10. The SEWAIR experiment; t =number of days, see Table 4 for the definition of x.

cells and the phase of planing off when the frequency of dms carrying cells stabilizes at around 90",,.

(2) The parameter values which produce these curves are biologically plausible. Due to chance the difference between the growth rates of the dms carrying cells and the dms free cells was the same in the two experiments, 4.7",,, though in vitro the cells without dms, and in vivo the cells with dms divided more rapidly. In the SEWAIR experiment the conversion of dms carrying cells into dms free cells is important, but even a very small value of m, 0.47"',, can produce a marked effect on the population. The value of m is of very little importance in the SEWAI case, but it is interesting to see that the value which fits the data in the best way is positive and not very far from the value of m obtained in the SEWAI R case. We see from the curves that it does not matter very much that m i s not depending on the composition of dms carrying cells. The effect of the conversion of cells becomes important only when there is a considerable proportion of dms carrying cells, and we know from Fig. 3 that the composition of the dms carrying cell population then is rather stabile. ( 3 ) An analysis has also been made of the SEWA4 and the SEWA7 experiments. I t turns out that a very good fit can be obtained between these observations and curves drawn with the same biologically relevant

parameter values as in the SEWAI experiment, i.e. CI = 0.047 and m = 0.0017. The only difference between the curves for the three in vitro experiments is, thus, that they "start" at different days. (4) A final word of warning should be included. We can not conclude from the good fit between the experiments and the simple model that the model is correct, only that it can give results which are consistent with the data. Completely different types of biological processes may produce the same final effect as the differential growth rates and the conversion of dms carrying cells into cells without dms, which we have included in our model. At present. however, the model discussed here seems to be the most plausible. Acknowledgments. Financial support of this work from the Swedish Cancer Society and from the John and Augusta Persson Foundation for Medical Research is gratefully acknowledged. -

Literature cited BIEDLER, J. L. and SPENGLER, B. A. 1976a. Metaphasechromosome anomaly: Association with drug resistance and cellspecific products. Science 191: 185 -187 B. A. 1976b. A novel chromoBIEDLER,J. L. and SPENGLER, some abnormality in human neuroblastoma and antifolateresistant Chinese hamster cell lines in culture. - J . Not. Cuncer Inst. 5 7 : 683-695 ~

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BILDLER.J . L.. HELSON. L. and SPENGLER, B. A . 1973. Morphology and growth. tumorigenicity. and cytogenetics of human neuroblastoma cells in continuous culture. ('unwr Research 33: 2643 2652 BIEI)I.ER.J. L.. ALRRECIIT'. A . M. and SPENOLER. B. A . 1974. Nonbanding (homogeneous) chromosome regions in cells Gencricr 77, with very high dihydrofolate reductase levels. Suppl. p. 4 s COX,D.. Y U N C K ~C. N . and SPRK;(;S, A . 1965. Minute chromatin bodies in malignant tumours of childhood. Luncrr (2):55--58 DONNER. L. and BUBENiK. J. 1968. Minute chromatin bodies in two mouse tumours induced in vivo by Rous Sarcoma Fuliu B i d . 14: R6 -88 virus. F t ~ ~ iE.j ,M., WIENER, F.. KLEIN.G. and HARRIS,H. 1973. Selection of tumor-host cell hybrids from polyoma virusJ . Nur. Cunand methylcholanthrene-induced sarcomas. w r Inst. 51: 1865 - 1875 G . 1974. The detailed chromosome constitution of a LEVAN. benzpyrene-induced rat sarcoma. A tentative model for G-band analysis in solid tumors. Hrrdirus 7X: 273 290 LEVAN.G. 1975. Cytogenetic studies in experimental rat The.sLs.Univ. Lund sarcomas. ~

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LEVAN, G. and LEVAN. A. 1975. Specific chromosome changes in malignancy: Studies in rat sarcomas induced by two Hrrcdirus 79: 161 I98 polycyclic hydrocarbons. U . , KLEIN,G and LEVAN.G., MANDAHI.,N., BREGULA, LEVAN,A . 1976. Double minute chromosomes are not centromeric regions of the host chromosomes. Nrredirus X3: 83-~90 LUBS, H. A. JR.. SALMON, J . H. and FLANIOAN, S. 1966. Studies of a glial tumor with multiple minute chromosomes. Cuncrr 19: 591--599 MARK,J. 1967. Double-minutes A chromosomal aberration Hurivfiru.\ 5 7 : 1-22 in Rous sarcomas in mice. MARK,J. 1970. Rous sarcomas in mice: The chromosomal progression during early in vivo transplantation. - Merediru.s 65: 59--82 N I E L S ~ NK. , 1976. Chromosomal evolution in the EhrlichLettre complex of hyperdiploid mouse ascites tumors: Herrdiro.s X4: Results from seven laboratory strains. 77--107 T.. HAYASHI, M., RABINOWITL, 2. and SACHS.L. YAMAMOTO, 1973. Chromosomal control of malignancy in tumors from cells transformed by polyoma virus. In!. J . Ctrncer 11: 555 - 566 ~~

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