Hrrediras 83 X3
YO ( I Y 7 6 )
Double minute chromosomes are not centromeric regions of the host chromosomes GORAN LEVAN, NILS MANDAHL, URSZULA BREGULA,' GEORGE KLEIN AND ALBERT LEVAN
Institute of' Gcwrtics, University af Lund Depurtment of' Tumor Biology, Karolinskrr Insfitutef,Stockholm, Sweden
LC.VAN. C.. MANDAHI., N., BREW( A. U., KLEIN. G. and LFVAN.A. 1976. Douhle minute chromoHrrrdiros 83: 83--90. Lund, somes are not centromeric regions of the host chromosomes, Sweden. ISSN 0018-0661. Received March 23. 1976 ~
The SEWA ascites tumor of the mouse contains, in addttlon to the ordinary chromosomes, a varying number of minute chromatin bodies, called double minutes (drns). Among 394 cells, 43 were without dms. the others had from I to 300 dms. The appearance of the drns suggested that they were acentric, which was also supported by the finding that even the largest dms, which were spheres with a diameter larger than the ordinary chromatid breadth. exhibited no C-heterochromatin in Hoechst 33258 fluorescence nor in C-banding with the BSG technique. The conclusion was that the dms of the SEWA tumor have not originated from centromeric regions of ordinary mouse chromosomes. Giirun L n u n , Insrirurc of Gmrrics. Unrivrsirj. of Ltord S-223 62 Lund, Swrdiw
The perplexing cytogenetic problem of the so-called double minute chromosomes (dms) their nature, origin, possible function - has been the subject of much speculation ever since they were first observed in human tumors by SPRIGCSet al. (1962) and in experimental tumors by MARK( 1967). So far everyone has agreed that they are of chromosomal nature, but exactly how they relate to ordinary chromosomes is still unknown. Their capacity to keep their foothold for long periods in malignant cell populations would indicate that they are provided with centromeres and thus either stem from the centromeric regions of normal chromosomes or have developed neocentric properties. In the latter case, they may have originated from any chromosomal region broken up into small fragments. Pictures of chromosome pulverization are often highly suggestive of dms formation, but recent results rather suggest that pulverization does not involve chromosome fragmentation ( ROHME 1975). Direct observation of the morphology of the dms has, however, often indicated that they are in fact acentric. So far, they have mostly been studied in ~
colchicinized cells, and during c-mitosis they appear as pairs of tiny spherical chromatin bodies, roughly keeping pace with the mitotic cycle of the cell. Their doubleness corresponds to the sister chromatids of ordinary chromosomes. During prophase and early metaphase the 2 halves of each dm stay close together, during metaphase and anaphase they separate, often widely. Even so, it is usually possible to determine which halves belong together, especially when there are size differences among the dms. 1t has been pointed out occasionally that dms of different size resemble acentric ring fragments (e.g. SANDBERG et al. 1972). It is quite clear, however, that minute chromosomes in the size order of the dms may have centromeric structures, and it is very likely that what has been referred to as dms may form a heterogeneous group of nuclear constituents. In a few cases, as in an RSV-induced mouse tumor (CBA283 in MARK1967) and in a likewise RSV-induced rat tumor (MITELMAN I On leave from the Department of Tumor Biology. Institute of Oncology, Warsaw. Poland
al. 1972) i t \\;is possible to associate the origin o f a number o f new dms uith the disappcar;incc ofthc w i n e number o f chromosomes from the host set. In ct
these ciiscs the dms were evidently centric and in the continuation showed good mitotic stability. This was also the c;ise with some of the dins i n the huniun testicular tumors reported by MAKTINITAI' ( 1966). in the human leukemias reported by PIERRI.et al. (1971) and in some of the BP-induced rat sarcomas reported by L W A Nand LEVAN(1975) not to mention the stable minute chroinosonies of serially carried ascites tuinors o f the mouse (BAYKI:U r t { i : K 1957: H A I ' S C H K A and LEVAN 1958). The distinction on rnorphologic criteria between centric and acentric dnis can be made only in the large ones. the small d m s being too near the limit of resolution to permit safe observations. In our present material. the SEWA-tumor. however. quite large dins are found. which i n every respect behave as the small ones and which are apparently without centromere. In view of the capacity o f dnis to remain in cell populations through considerable periods ;is far as we knou. permanently - it is undoubtedly controversial t o assume that they can exist without functioning centromeres. Thus. a n idea for their origin. widely accepted and expressed, together with other possibilities, already by M A R Kin his pioneer paper o f 1967. claims that the dms are cut-off centromeric regions o f ordinary chrornosomes. This idea can now be put t o the test thanks to the new banding techniques which include those that specificnllq stain the centromeric heterochromatin in certain species. By this means a safe distinction can be made hetween the neighborhood o f the centroinere and other parts of the chrornosnme body. The species o f choice for this purpose is the mouse, and for some time we have been o n the look-out for a mouse tumor with ;I high, regular incidence of dms. preferably of large enough sire to make it possible to decide whether they stain a s C-heterochromatin or not. Recently. it was found that the SEWA tumor, widely used in experimental oncologic work, fulfilled these conditions. and the present communication is a report on the responses o f the SEWA dms t o a couple of specitic C-heterochromatin stainings.
Material and methods The SEWA tumor was induced by polyomu virus i n an A.SW mouse (SJOGREN et al. 1961). I t was originally an osteogenic sarcoma hut has lost its osteogenic differentiation (KLI;IN et al. 1971). I t
conwrted i n t o ascitic form by NOKDIssKJiji 1) The ;iscites tumor is maintained hy serial tr;iiispl~iint~itioii in A.SW mice. M hich u i l l h e lor 3- 4 weeks after ;in inoculum of 10" cells. SEWA was one of the piircntnl strains i n the ti! bridi/ation u o r k of Wi1.st.K and coll;ihoratorb. summari/cd in W1t.w.K ( 1974). I h r i n g this work the chromosomes were studied currently. The chromosome number formed a narrow mode at 43. Fig. 2a in FmYO et ;iI. (1973, p. 1874). s h w i n g a c-inetaphase of SEWA with 43 chromosonics, is especially interesting in the present context, since i t contains a great many dins of different sizes. I n the text the SEWA chromosomes were described, a s follow~s:"All chromosomes were acrocentric. subtelocentric. or telocentric. The following marker5 were present: an abnormally long telocentric chroniosome. a chromosome with a secondary constriction. and se\er;iI minute chromosomes o r fragments" (1.c.. p. 1x67). In the present work, passages 46 and 48 of the same SEWA line were used ;is in the work o f b.i.sy0 and collaborators. The chroinosome study was undertaken in the following a a y : Dry spreads were first stained in Giemsa and inetaphases drawn and photographed. The frequency o f cells ~ i t dim. h the number of dins per cell and their size and appearance were determined. After removal of the immersion oil with xylene. destaining in methanol- acetic acid and drying, the slides were stained in "Hoechst 333%" according t o LAI-I (1073) and mounted in glycerol. The cells prebiously drawn a n d photographed in Giemsa were now restudied and photographed in the Iluorescence microscope. Next. the glycerol was removed. the slides were taken back to methanol acetic acid. dried and processed according to the HSG technique (St.siNw 1972). slightly inodilied ( M A N I I A I I Iand . FKI.I)GA 1075). Again the same cells. their chromos o m e s no\\ showing C-hands. \\ere photographed. These rather complex and exacting procedures yielded ii rich material of pictures showing the same cells under 3 dil'ferent aspects: ( I ) plain Giemsa. ( 2 ) Hoechst 33358 Iluorescence. and ( 3 ) C-handing. The photography o f the dnis posed considerable problems because of the large difference in size betMeen the ordinary chromosomes and the average dins. In order to make the latter visible i n the photographs it u a s often necessary to overexpose the negatives and t o exaggerate the printing, uith the result that the ordinary chromosomes lost in detail. \\;is
( 1964).
DOUR1 E MlhU IrS AN11 HOST CHKOMOSOMIS
85
40
30
20
1c
Fig I. I h t r i h u t i o i i o l t h e nuinbcrs oldrnh in 3Y4 SEWA cells
Observations I . Ordinary chromosomes The stemline karyotype of the ordinary chromosomes was in good agreement with the observations of FirNYii et al. (1973). The stemline chromosome number was S = 4 3 and the second most common number was 44.90",, of the total stemline population having S + I c h r o m o ~ o m e(Table ~ I ) . Cells in the double stemline region were few; in 1 slide. scanned for polyploid metaphases, 4 among 193 (2.l",,) were hypertetraploid with 86& 88 chromosomes. The long marker chromosome with a secondary constriction mentioned by F E N Yet~ al. (1973) was seen in alniost every cell.
2. Incidence of the dms The dms were counted, with the aid of the drawing mirror, in 394 cells of 5 slides. Their numbers are represented in the diagram of Fig. 1. All except 43 cells had at least I dm. The distribution of dms per cell was wide. the highest number counted in I cell with fair accuracy was 300 dins. thus 600 discrete bodies, but we do not doubt that there were cells with much higher numbers that could not be determined accurately. In SEWA cells in culture we have counted well above 1000 dms in one cell: the cell of Fig. 2 b from such a culture has approximately SO0 dms. As seen from Fig. I , there is disregarding the zero class - an indication of a flat mode from about 3 to 25 drns and. perhaps a second, lower mode at 40 to 60 dms. The distribution is gradually tapering off upwards from the modal region and is ~
a Fig 2
b .I
a n d h S E W A cell\ n i t h \\idel) differcnt nun1hers o f d n i \ .
:I'
17: h . around 5 0 0 dni\ from
3
cell in culture.
paired dms were more frequent in cells, in which the ordinary chromosomes were at prophase or prometaphase, whereas the proportion of unpaired dms increased with advancing metaphase- anaphase. In the cell of Fig. 2 a, which is a fairly early metaphase, all 17 dms were strictly paired. whereas in Fig. 2 b, which represents :I more advanced stage. the daughter halves of the dms were at some distance from each other; still there was usually no difficulty to see which halves belonged together. We have been able so far to study the dms only in colchicine- and hypotonic-treated cells. thus during c-mitosis. In spite o f several attempts to analyze them during normal mitosis, we have failed to obtain a clear picture o f their behavior on the mitotic spindle. So much can be said, however, that during normal metaphase. when the ordinary chromosomes of the mouse usually arrange themselves
fairly continuous up till well above 100 dms per cell. Above 1 15 there were only Y counts: 128, 134, 143, 169. 184, 205, 217. 232 and 300. The high number of cells without dms may indicate that once the dms are lost from a cell the progeny from this cell will continue having no dms. whereas those with at least I o r a few dms may undergo both decrease and increase of dms in their progeny. I t appears from the diagram that the distribution fits neither a normal, nor a Poisson distribution. 3. Shape and size of the drns The dins of the SEWA tumor had the shape characteristic of most of these peculiar small bodies so far reported; they appeared in pairs. and the distance between the members of each pair was highly variable. As mentioned in the introduction. closely
T d h I. Distribution of chromosome numbers in 5 slides of the SEWA tumor Chromowrne nun1her:
39
Number o f c e l l s : 3 Percentage: 0.8
40
4 10
41
42
43
44
I2 3.1
37 Y.5
210 S3.7
I06
IS
1
27.1
3.8
0.5
45
40
47
48
Total Mean
I 0.3
I 0.3
100.1
301 43 17
DOUBLE MINUTES AND HOST CHROMOSOMES
Herrdirus 83 (1976)
87
..
Fig. 3 a--d. SEWA cell with 43 chromosomes and 76 dms o f different sizes; a, b: no specific treatment; c: Hoechst 33258; d: C-banding. Numerals I lo S indicate the biggest dms; it is evident in c and d that even the big dms d o not show C-heterochromatin. In a. the arrow indicates the marker with secondary constriction; the interstitial C-band at the constriction is visible in c and d . x 1530.
into a ring with their centromeres turned inwards, the dms are never or rarely located inside the ring, as would have been expected in case they were centric. Instead they are usually found near to or in direct contact with the outer surface of the ring, often in groups or in single rows. In normal anaphases, the dms are almost never seen. During telophase and interphase, very small micronuclei occur. Their frequency is much lower than would be expected if the dms regularly formed micronuclei. In one fixation we counted from 5 to 15% cells with 1 to 5 micronuclei. They often stained very faintly and looked as if they were disintegrating.
The dms of SEWA vary in size much more than is usual among dms. The smallest ones are barely visible, often just perceivable as a faint marbling of the background. Usually they are small rounded double dots, their diameter being only a small fraction of that of the chromatids of the ordinary chromosomes of the same cell. Much larger dms are also found, the largest being equal to or even larger than the diameter of the chromatids (Fig. 3). The largest ones are usually in minority in the cell and found together with many small ones. Irrespective of their size, the biggest dms have the same shape and behavior as the small ones. The heterogeneity
88
G . LEVAN I T A L
#-
I
a
C
w
d Fig. 4 a I . Parts 013 SEWA cells: ;I. c. u: (;lcm\a: h, d. I wine cell\ after C'-h;inding. Although the c~.ntroiiicric heterochromatin 1 5 dried darkly in the Iattcr pictures. the dins ;ire \taincd \cr) l a i n t l ~ ~ . n d i c a t ~ nt hga t they arc n o t ('-hclerochrom;itic. Thc x r o u s i n c I indic;ite the markcr uith \ccoi~d;ir) constriction. x 1x50
of the dms population is surprising. Although there are many cells with apparently equal-sized dms. the over-all size level of dms may vary considerably among cells. This would be understandable if the total pool of dms in a cell would gradually increase in size, but would be hard to reconcile with the assumption of stable clones perpetuating this multiformity. The dms of each clone may have the .capacity of varying not only in number but also in size. 4. C-hand staining
The main purpose of the present investigation was to decide whether the dms stained as C-heterochromatin or not. A considerable number of metaphases in 2 slides were photographed in Giemsa before any
special treatment and then in fluorescence and eventually in C-banding as described in the material and method section above. The result of these procedures in one cell is shown in Fig. 3, in which a is a drawing and b a photograph in ordinary Gienisa; c is a Hoechst 33258 staining and d a C-banding. The cell has 43 ordinary chromosomes, among them the big marker with ;1 constriction. described by F I . N Yet~ ~al. (1973). This marker is indicated by an iirrow in Fig. 3 a. I t is seen in c and d that this marker has an interstitial C-band at the site of the constriction. The cell also had 76 dms of different sizes. Among them. 3 single and 2 double. marked with the numbers 1 t o 5 in the figure. were much larger than the rest. In the original staining, from which the drawing of Fig. 3 a was made, at least No. I t o 4 appeared thicker than the ordinary
chromatid diameter. In Fig. 3 b, after theexaggerated exposure in printing, the chromosomes appeared thicker than in reality. I t should be noted that the big dms all through this investigation were spherical in shape and exhibited no signs of being centric. In the paired ones, No. 4 and 5. the sister halves were in contact with each other at one point but showed no connecting fiber. Also No. 1 and 2, which were quite remote from each other, had probably belonged to one pair originally and thus were sister halves. No. 3 had no mate, which was exceptional; it may have been single from the beginning, but more likely its sister half had been lost at the flattening of the cell. The biggest ones, No. 1 and 2 , undoubtedly were suggestive of ring shape, although no openings of the rings were perceptible. The rest of the dms were all smaller, and although there were quite striking size differences among them, the contrast was rather great between Nos. 1 to 5 , on the one hand, and the largest ones of the rest, on the other hand. Fig. 3 c shows all the ordinary chromosomes with light proximal C-heterochromatin. With this staining only faint traces were visible of the largest dms, indicating that the dms are not C-heterochromatic. A similar result was obtained with the C-banding of Fig. 3d: the centromeric regions of the ordinary chromosomes were stained heavily, but the dms were barely visible. There is no doubt that the dms stained more like chromosome arms than centromeric regions. In the 2 preparations treated in this way, some 30 cells were photographed and analyzed, as just described with Fig. 3. After C-staining they wereall carefully rechecked in the microscope and all gave the same result: the dms were generally more faintly stained than even the most faintly appearing regions of the chromosome arms. In fig. 4 parts of 3 cells have been displayed in Giemsa (a. c. e) and in C-banding (b, d, f). The big marker with interstitial C-heterochromatin is indicated by arrows in d and f. The 3 cells of Fig. 4 behaved in the same manner. clearly indicating that the dms were not C-heterochromatic. Only in very exceptional cases, once or twice in the entire material, it happened that a structure that had been classed as belonging to the dms became heavily stained in C-banding. On closer analysis these structures turned out to be small chromosomes, clearly centric and of a different shape from the dms. In summary. it can be stated that the present analysis gave conclusive evidence that the dms of the SEWA tumor have not been derived from unchanged centromeric regions of the mouse chromosomes.
Discussion A fundamental question for the understanding of the nature of the dms is whether they are provided with a centromere or not. The fact that they behave as chromosomes during mitosis and have the capacity to remain for long periods in populations would a priori indicate that they are centric. Conversely. the fact ascertained in the present investigation that the SEWA dms do not originate from centromeric regions of the chromosomes of the host species speaks against their being centric. Even though mechanisms can perhaps be perceived, by which a C-banded chromosome segment may loose its Cheterochromatic condition, this is a possibility that seems far-fetched. The fact that minute chromosomes exist that certainly are centric makes it necessary to maintain the distinction between centric and acentric minutes. Since the former group includes many different kinds of chromosomes: minute chromosomes in natural species of mosses, higher plants. insects, birds. mammals etc., the so-called accessory or B-chromosomes in many materials of both plants and animals, and also minutes with C-heterochromatin in malignant cells and cell lines (for instance SHEPARD et al. 1974; HHNEEN1976; N I I L S ~ N 1976). it seems convenient to restrict the term of dins to the latter group. which has been found so far only in malignant cells. Also, it should be remembered that the SEWA tumor with its apparently acentric dms is just one instance: it may well be that dms even in the restricted sense may include centric structures in other materials. When we assume that dnis are really acentric. the question immediately arises: how is it possible that acentric chromosomes can remain in the population through long periods? Acentric chromatin bodies would normally fail to be ihcludcd into the telophase nuclei and become eliminated. One possibility would be that dms are formed de novo during each mitotic cycle. Their appearance accords well with that of pairs of small acentric rings. This mechanism would require a specific instability of the chromosomes during GI leading to the punching out of small loops of the DNA thread which would form pairs of acentrics during next mitosis. This mechanism would lead to gradual reduction in length of the chromosomes and is fairly unlikely also for other reasons. Another. so far purely theoretic possibility. should be mentioned: The heritable constituent of the dms may be of viral origin rather than of chromosomal. Like most of the dms-carrying experimental tumors the SEWA has viral etiology; it was induced originally by polyoma virus and still carries the polyoma-
90
Heredirus 83 (1976)
G . LEVAN ET AL.
specific transplantation antigen (KLEINet a[. 1971; KLEINunpubl.). It has been shown recently that chromatin-like structures, “minichromosomes”, have been formed both in vivo and in vitro by SV40 and polyoma DNA after association with histones (GRIFFITH 1975; GERMOND et a]. 1975; CREMISI et al. 1976). The possible origin of dms from viral DNA has some attractive points, and experiments are being planned to test this possibility.
LATT,S. A . 1973. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. - Proc. Nur. Acud. Sci. 7 0 : 3385 3399 LEVAN. G. and LEVAN. A. 1975. Specific chromosome changes in malignancy: Studies in rat sarcomas induced by two polycyclic hydrocarbons. Hereditas 7Y: 161 . - 198 MANDAHL. N. and FREDC~A. K. 1975. Q-, G-, and C-band patterns of the mink chromosomes. - Heredirus 81: 21 1-220 MARK.J . 1967. Double-minutes. A chromosomal aberration in Rous sarcomas in mice. - Hereditus 5 7 : I -22 MARTINEAU. M. 1966. A similar marker chromosome in testicular tumours. Luncet ( I ) : 839% 842 F.. LEVAN. G . and MARK,J. 1972. The origin of MITELMAN, Acro Parhol. Micro-double-minutes in Rous rat-sarcoma. h i . Scand. A CIO: 428-429 NIELS~N K., 1976. A near-hexaploid Ehrlich-Lettre mouse ascites tumor line with low sensitivity to colchicine. HeriditusXJ: 105- 122 NORDENSKJBLD, B. 1964. Large scale production of polyoma virus in mouse ascites tumor cells in vivo. - Virology 24: 225- 227 C. and LINMAN, J. 1971. MicrochroPIERRE. R.. HOAGLAND. Cancer mosomes in human preleukemia and leukemia. 27: 160- 175 ROHME.D. 1975. Evidence suggesting chromosome continuity during the S-phase of Indian muntjac cells. - Heredirus 80: 145 149 SANDBERG, A. A,, SAKURAI, M. and HOLDSWORTH. R. 1972. Chromosomes and causation of human cancer and leukemia. VIII. DMSchromosomes in a neuroblastoma. - Cuncer 29: 1671-1679 J. S., WURSTER-HILL, D. H., PETTENGILL, 0. S. SHEPARD, and SORENStN. G. D. 1974. Giemsa-banded chromosomes of mouse myeloma in relationship to oncogenicity. Cytogiwet. Cell Genet. 13: 279-309 S ~ f f i ~H.t ~0.. , HELLSTRAM, I . and KLEIN.G. 1961. Transplantation of polyoma virus induced tumors in mice. C m w r Res. 2 i : 329 -337 M. M. ~ ~ ~ C L A R C.KM.E 1962. . SPRIGGS. A. 1.. BODDINGTON. Chromosomes of human cancer cells. Brrr. Med. J . ( 2 ) : 1431 -1435 SUMNER, A . T. 1972. A simple technique for demonstrating . Re.7. 75: 304-306 centromeric heterochromatin. - E . Y ~Cell WIENER,F. 1974. Studies on the chromosomal control of malignant behaviour by cell hybridization. - Thesis. Univ. Stockholm -
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Acknouledgments. - 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 BAYREUTHER, K. 1952. Der Chromosomenbestand des EhrlichAscites-Tumors der Maus. Z . Nururforsch. 7 h: 544-557 CREMISI, C., PIGNATTI. P. F., CROISSANT. 0. and YANIV.M. 1976. Chromatin-like structures in polyoma virus and simian J . Virol. 17: 204 -21 I virus 40 lytic cycle. FENYO.E. M.. WIENER.F.. KLEIN,G. and HARRIS,H. 1973. Selection of tumor-host cell hybrids from polyoma virusJ . Nut. (’onand methylcholanthrene-induced sarcomas. cer Insr. 5I: 1865-1875 J . E., HIRT. B., OUDET,P., GROSS-BELLARD. M. GERMOND, and CHAMBON. P. 1975. Folding of the DNA double helix in chromatin-like structures from simian virus 40. - Proc. Nar. Ai,ad. Scr. 72: lX43--1847 GRIFFITH,J. D. 1975. Chromatin structure: Deduced from a minichromosome. - Science 187: 1202- 1203 HAUSCHKA, T. S. and LEVAN, A. 1958. Cytologic and functional characterization of single cell clones isolated from the J . Nut. (’uncer Insr. Krebs-2 and Ehrlich ascites tumors. 21: 77-135 HENEEN, W. K 1976. HeLa cells and their possible contamination of other cell lines: Karyotype studies. - Hereditus X2: 217-248 U . , WIFNER,F. and HARRIS,H. 1971. KLEIN,G.. BRFGULA, The analysis of malignancy by cell fusion. I . Hybrids between J . Cell Sci. 8 : tumour cells and L cell derivatives. 659-672 ~~
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YO ( I Y 7 6 )
Double minute chromosomes are not centromeric regions of the host chromosomes GORAN LEVAN, NILS MANDAHL, URSZULA BREGULA,' GEORGE KLEIN AND ALBERT LEVAN
Institute of' Gcwrtics, University af Lund Depurtment of' Tumor Biology, Karolinskrr Insfitutef,Stockholm, Sweden
LC.VAN. C.. MANDAHI., N., BREW( A. U., KLEIN. G. and LFVAN.A. 1976. Douhle minute chromoHrrrdiros 83: 83--90. Lund, somes are not centromeric regions of the host chromosomes, Sweden. ISSN 0018-0661. Received March 23. 1976 ~
The SEWA ascites tumor of the mouse contains, in addttlon to the ordinary chromosomes, a varying number of minute chromatin bodies, called double minutes (drns). Among 394 cells, 43 were without dms. the others had from I to 300 dms. The appearance of the drns suggested that they were acentric, which was also supported by the finding that even the largest dms, which were spheres with a diameter larger than the ordinary chromatid breadth. exhibited no C-heterochromatin in Hoechst 33258 fluorescence nor in C-banding with the BSG technique. The conclusion was that the dms of the SEWA tumor have not originated from centromeric regions of ordinary mouse chromosomes. Giirun L n u n , Insrirurc of Gmrrics. Unrivrsirj. of Ltord S-223 62 Lund, Swrdiw
The perplexing cytogenetic problem of the so-called double minute chromosomes (dms) their nature, origin, possible function - has been the subject of much speculation ever since they were first observed in human tumors by SPRIGCSet al. (1962) and in experimental tumors by MARK( 1967). So far everyone has agreed that they are of chromosomal nature, but exactly how they relate to ordinary chromosomes is still unknown. Their capacity to keep their foothold for long periods in malignant cell populations would indicate that they are provided with centromeres and thus either stem from the centromeric regions of normal chromosomes or have developed neocentric properties. In the latter case, they may have originated from any chromosomal region broken up into small fragments. Pictures of chromosome pulverization are often highly suggestive of dms formation, but recent results rather suggest that pulverization does not involve chromosome fragmentation ( ROHME 1975). Direct observation of the morphology of the dms has, however, often indicated that they are in fact acentric. So far, they have mostly been studied in ~
colchicinized cells, and during c-mitosis they appear as pairs of tiny spherical chromatin bodies, roughly keeping pace with the mitotic cycle of the cell. Their doubleness corresponds to the sister chromatids of ordinary chromosomes. During prophase and early metaphase the 2 halves of each dm stay close together, during metaphase and anaphase they separate, often widely. Even so, it is usually possible to determine which halves belong together, especially when there are size differences among the dms. 1t has been pointed out occasionally that dms of different size resemble acentric ring fragments (e.g. SANDBERG et al. 1972). It is quite clear, however, that minute chromosomes in the size order of the dms may have centromeric structures, and it is very likely that what has been referred to as dms may form a heterogeneous group of nuclear constituents. In a few cases, as in an RSV-induced mouse tumor (CBA283 in MARK1967) and in a likewise RSV-induced rat tumor (MITELMAN I On leave from the Department of Tumor Biology. Institute of Oncology, Warsaw. Poland
al. 1972) i t \\;is possible to associate the origin o f a number o f new dms uith the disappcar;incc ofthc w i n e number o f chromosomes from the host set. In ct
these ciiscs the dms were evidently centric and in the continuation showed good mitotic stability. This was also the c;ise with some of the dins i n the huniun testicular tumors reported by MAKTINITAI' ( 1966). in the human leukemias reported by PIERRI.et al. (1971) and in some of the BP-induced rat sarcomas reported by L W A Nand LEVAN(1975) not to mention the stable minute chroinosonies of serially carried ascites tuinors o f the mouse (BAYKI:U r t { i : K 1957: H A I ' S C H K A and LEVAN 1958). The distinction on rnorphologic criteria between centric and acentric dnis can be made only in the large ones. the small d m s being too near the limit of resolution to permit safe observations. In our present material. the SEWA-tumor. however. quite large dins are found. which i n every respect behave as the small ones and which are apparently without centromere. In view of the capacity o f dnis to remain in cell populations through considerable periods ;is far as we knou. permanently - it is undoubtedly controversial t o assume that they can exist without functioning centromeres. Thus. a n idea for their origin. widely accepted and expressed, together with other possibilities, already by M A R Kin his pioneer paper o f 1967. claims that the dms are cut-off centromeric regions o f ordinary chrornosomes. This idea can now be put t o the test thanks to the new banding techniques which include those that specificnllq stain the centromeric heterochromatin in certain species. By this means a safe distinction can be made hetween the neighborhood o f the centroinere and other parts of the chrornosnme body. The species o f choice for this purpose is the mouse, and for some time we have been o n the look-out for a mouse tumor with ;I high, regular incidence of dms. preferably of large enough sire to make it possible to decide whether they stain a s C-heterochromatin or not. Recently. it was found that the SEWA tumor, widely used in experimental oncologic work, fulfilled these conditions. and the present communication is a report on the responses o f the SEWA dms t o a couple of specitic C-heterochromatin stainings.
Material and methods The SEWA tumor was induced by polyomu virus i n an A.SW mouse (SJOGREN et al. 1961). I t was originally an osteogenic sarcoma hut has lost its osteogenic differentiation (KLI;IN et al. 1971). I t
conwrted i n t o ascitic form by NOKDIssKJiji 1) The ;iscites tumor is maintained hy serial tr;iiispl~iint~itioii in A.SW mice. M hich u i l l h e lor 3- 4 weeks after ;in inoculum of 10" cells. SEWA was one of the piircntnl strains i n the ti! bridi/ation u o r k of Wi1.st.K and coll;ihoratorb. summari/cd in W1t.w.K ( 1974). I h r i n g this work the chromosomes were studied currently. The chromosome number formed a narrow mode at 43. Fig. 2a in FmYO et ;iI. (1973, p. 1874). s h w i n g a c-inetaphase of SEWA with 43 chromosonics, is especially interesting in the present context, since i t contains a great many dins of different sizes. I n the text the SEWA chromosomes were described, a s follow~s:"All chromosomes were acrocentric. subtelocentric. or telocentric. The following marker5 were present: an abnormally long telocentric chroniosome. a chromosome with a secondary constriction. and se\er;iI minute chromosomes o r fragments" (1.c.. p. 1x67). In the present work, passages 46 and 48 of the same SEWA line were used ;is in the work o f b.i.sy0 and collaborators. The chroinosome study was undertaken in the following a a y : Dry spreads were first stained in Giemsa and inetaphases drawn and photographed. The frequency o f cells ~ i t dim. h the number of dins per cell and their size and appearance were determined. After removal of the immersion oil with xylene. destaining in methanol- acetic acid and drying, the slides were stained in "Hoechst 333%" according t o LAI-I (1073) and mounted in glycerol. The cells prebiously drawn a n d photographed in Giemsa were now restudied and photographed in the Iluorescence microscope. Next. the glycerol was removed. the slides were taken back to methanol acetic acid. dried and processed according to the HSG technique (St.siNw 1972). slightly inodilied ( M A N I I A I I Iand . FKI.I)GA 1075). Again the same cells. their chromos o m e s no\\ showing C-hands. \\ere photographed. These rather complex and exacting procedures yielded ii rich material of pictures showing the same cells under 3 dil'ferent aspects: ( I ) plain Giemsa. ( 2 ) Hoechst 33358 Iluorescence. and ( 3 ) C-handing. The photography o f the dnis posed considerable problems because of the large difference in size betMeen the ordinary chromosomes and the average dins. In order to make the latter visible i n the photographs it u a s often necessary to overexpose the negatives and t o exaggerate the printing, uith the result that the ordinary chromosomes lost in detail. \\;is
( 1964).
DOUR1 E MlhU IrS AN11 HOST CHKOMOSOMIS
85
40
30
20
1c
Fig I. I h t r i h u t i o i i o l t h e nuinbcrs oldrnh in 3Y4 SEWA cells
Observations I . Ordinary chromosomes The stemline karyotype of the ordinary chromosomes was in good agreement with the observations of FirNYii et al. (1973). The stemline chromosome number was S = 4 3 and the second most common number was 44.90",, of the total stemline population having S + I c h r o m o ~ o m e(Table ~ I ) . Cells in the double stemline region were few; in 1 slide. scanned for polyploid metaphases, 4 among 193 (2.l",,) were hypertetraploid with 86& 88 chromosomes. The long marker chromosome with a secondary constriction mentioned by F E N Yet~ al. (1973) was seen in alniost every cell.
2. Incidence of the dms The dms were counted, with the aid of the drawing mirror, in 394 cells of 5 slides. Their numbers are represented in the diagram of Fig. 1. All except 43 cells had at least I dm. The distribution of dms per cell was wide. the highest number counted in I cell with fair accuracy was 300 dins. thus 600 discrete bodies, but we do not doubt that there were cells with much higher numbers that could not be determined accurately. In SEWA cells in culture we have counted well above 1000 dms in one cell: the cell of Fig. 2 b from such a culture has approximately SO0 dms. As seen from Fig. I , there is disregarding the zero class - an indication of a flat mode from about 3 to 25 drns and. perhaps a second, lower mode at 40 to 60 dms. The distribution is gradually tapering off upwards from the modal region and is ~
a Fig 2
b .I
a n d h S E W A cell\ n i t h \\idel) differcnt nun1hers o f d n i \ .
:I'
17: h . around 5 0 0 dni\ from
3
cell in culture.
paired dms were more frequent in cells, in which the ordinary chromosomes were at prophase or prometaphase, whereas the proportion of unpaired dms increased with advancing metaphase- anaphase. In the cell of Fig. 2 a, which is a fairly early metaphase, all 17 dms were strictly paired. whereas in Fig. 2 b, which represents :I more advanced stage. the daughter halves of the dms were at some distance from each other; still there was usually no difficulty to see which halves belonged together. We have been able so far to study the dms only in colchicine- and hypotonic-treated cells. thus during c-mitosis. In spite o f several attempts to analyze them during normal mitosis, we have failed to obtain a clear picture o f their behavior on the mitotic spindle. So much can be said, however, that during normal metaphase. when the ordinary chromosomes of the mouse usually arrange themselves
fairly continuous up till well above 100 dms per cell. Above 1 15 there were only Y counts: 128, 134, 143, 169. 184, 205, 217. 232 and 300. The high number of cells without dms may indicate that once the dms are lost from a cell the progeny from this cell will continue having no dms. whereas those with at least I o r a few dms may undergo both decrease and increase of dms in their progeny. I t appears from the diagram that the distribution fits neither a normal, nor a Poisson distribution. 3. Shape and size of the drns The dins of the SEWA tumor had the shape characteristic of most of these peculiar small bodies so far reported; they appeared in pairs. and the distance between the members of each pair was highly variable. As mentioned in the introduction. closely
T d h I. Distribution of chromosome numbers in 5 slides of the SEWA tumor Chromowrne nun1her:
39
Number o f c e l l s : 3 Percentage: 0.8
40
4 10
41
42
43
44
I2 3.1
37 Y.5
210 S3.7
I06
IS
1
27.1
3.8
0.5
45
40
47
48
Total Mean
I 0.3
I 0.3
100.1
301 43 17
DOUBLE MINUTES AND HOST CHROMOSOMES
Herrdirus 83 (1976)
87
..
Fig. 3 a--d. SEWA cell with 43 chromosomes and 76 dms o f different sizes; a, b: no specific treatment; c: Hoechst 33258; d: C-banding. Numerals I lo S indicate the biggest dms; it is evident in c and d that even the big dms d o not show C-heterochromatin. In a. the arrow indicates the marker with secondary constriction; the interstitial C-band at the constriction is visible in c and d . x 1530.
into a ring with their centromeres turned inwards, the dms are never or rarely located inside the ring, as would have been expected in case they were centric. Instead they are usually found near to or in direct contact with the outer surface of the ring, often in groups or in single rows. In normal anaphases, the dms are almost never seen. During telophase and interphase, very small micronuclei occur. Their frequency is much lower than would be expected if the dms regularly formed micronuclei. In one fixation we counted from 5 to 15% cells with 1 to 5 micronuclei. They often stained very faintly and looked as if they were disintegrating.
The dms of SEWA vary in size much more than is usual among dms. The smallest ones are barely visible, often just perceivable as a faint marbling of the background. Usually they are small rounded double dots, their diameter being only a small fraction of that of the chromatids of the ordinary chromosomes of the same cell. Much larger dms are also found, the largest being equal to or even larger than the diameter of the chromatids (Fig. 3). The largest ones are usually in minority in the cell and found together with many small ones. Irrespective of their size, the biggest dms have the same shape and behavior as the small ones. The heterogeneity
88
G . LEVAN I T A L
#-
I
a
C
w
d Fig. 4 a I . Parts 013 SEWA cells: ;I. c. u: (;lcm\a: h, d. I wine cell\ after C'-h;inding. Although the c~.ntroiiicric heterochromatin 1 5 dried darkly in the Iattcr pictures. the dins ;ire \taincd \cr) l a i n t l ~ ~ . n d i c a t ~ nt hga t they arc n o t ('-hclerochrom;itic. Thc x r o u s i n c I indic;ite the markcr uith \ccoi~d;ir) constriction. x 1x50
of the dms population is surprising. Although there are many cells with apparently equal-sized dms. the over-all size level of dms may vary considerably among cells. This would be understandable if the total pool of dms in a cell would gradually increase in size, but would be hard to reconcile with the assumption of stable clones perpetuating this multiformity. The dms of each clone may have the .capacity of varying not only in number but also in size. 4. C-hand staining
The main purpose of the present investigation was to decide whether the dms stained as C-heterochromatin or not. A considerable number of metaphases in 2 slides were photographed in Giemsa before any
special treatment and then in fluorescence and eventually in C-banding as described in the material and method section above. The result of these procedures in one cell is shown in Fig. 3, in which a is a drawing and b a photograph in ordinary Gienisa; c is a Hoechst 33258 staining and d a C-banding. The cell has 43 ordinary chromosomes, among them the big marker with ;1 constriction. described by F I . N Yet~ ~al. (1973). This marker is indicated by an iirrow in Fig. 3 a. I t is seen in c and d that this marker has an interstitial C-band at the site of the constriction. The cell also had 76 dms of different sizes. Among them. 3 single and 2 double. marked with the numbers 1 t o 5 in the figure. were much larger than the rest. In the original staining, from which the drawing of Fig. 3 a was made, at least No. I t o 4 appeared thicker than the ordinary
chromatid diameter. In Fig. 3 b, after theexaggerated exposure in printing, the chromosomes appeared thicker than in reality. I t should be noted that the big dms all through this investigation were spherical in shape and exhibited no signs of being centric. In the paired ones, No. 4 and 5. the sister halves were in contact with each other at one point but showed no connecting fiber. Also No. 1 and 2, which were quite remote from each other, had probably belonged to one pair originally and thus were sister halves. No. 3 had no mate, which was exceptional; it may have been single from the beginning, but more likely its sister half had been lost at the flattening of the cell. The biggest ones, No. 1 and 2 , undoubtedly were suggestive of ring shape, although no openings of the rings were perceptible. The rest of the dms were all smaller, and although there were quite striking size differences among them, the contrast was rather great between Nos. 1 to 5 , on the one hand, and the largest ones of the rest, on the other hand. Fig. 3 c shows all the ordinary chromosomes with light proximal C-heterochromatin. With this staining only faint traces were visible of the largest dms, indicating that the dms are not C-heterochromatic. A similar result was obtained with the C-banding of Fig. 3d: the centromeric regions of the ordinary chromosomes were stained heavily, but the dms were barely visible. There is no doubt that the dms stained more like chromosome arms than centromeric regions. In the 2 preparations treated in this way, some 30 cells were photographed and analyzed, as just described with Fig. 3. After C-staining they wereall carefully rechecked in the microscope and all gave the same result: the dms were generally more faintly stained than even the most faintly appearing regions of the chromosome arms. In fig. 4 parts of 3 cells have been displayed in Giemsa (a. c. e) and in C-banding (b, d, f). The big marker with interstitial C-heterochromatin is indicated by arrows in d and f. The 3 cells of Fig. 4 behaved in the same manner. clearly indicating that the dms were not C-heterochromatic. Only in very exceptional cases, once or twice in the entire material, it happened that a structure that had been classed as belonging to the dms became heavily stained in C-banding. On closer analysis these structures turned out to be small chromosomes, clearly centric and of a different shape from the dms. In summary. it can be stated that the present analysis gave conclusive evidence that the dms of the SEWA tumor have not been derived from unchanged centromeric regions of the mouse chromosomes.
Discussion A fundamental question for the understanding of the nature of the dms is whether they are provided with a centromere or not. The fact that they behave as chromosomes during mitosis and have the capacity to remain for long periods in populations would a priori indicate that they are centric. Conversely. the fact ascertained in the present investigation that the SEWA dms do not originate from centromeric regions of the chromosomes of the host species speaks against their being centric. Even though mechanisms can perhaps be perceived, by which a C-banded chromosome segment may loose its Cheterochromatic condition, this is a possibility that seems far-fetched. The fact that minute chromosomes exist that certainly are centric makes it necessary to maintain the distinction between centric and acentric minutes. Since the former group includes many different kinds of chromosomes: minute chromosomes in natural species of mosses, higher plants. insects, birds. mammals etc., the so-called accessory or B-chromosomes in many materials of both plants and animals, and also minutes with C-heterochromatin in malignant cells and cell lines (for instance SHEPARD et al. 1974; HHNEEN1976; N I I L S ~ N 1976). it seems convenient to restrict the term of dins to the latter group. which has been found so far only in malignant cells. Also, it should be remembered that the SEWA tumor with its apparently acentric dms is just one instance: it may well be that dms even in the restricted sense may include centric structures in other materials. When we assume that dnis are really acentric. the question immediately arises: how is it possible that acentric chromosomes can remain in the population through long periods? Acentric chromatin bodies would normally fail to be ihcludcd into the telophase nuclei and become eliminated. One possibility would be that dms are formed de novo during each mitotic cycle. Their appearance accords well with that of pairs of small acentric rings. This mechanism would require a specific instability of the chromosomes during GI leading to the punching out of small loops of the DNA thread which would form pairs of acentrics during next mitosis. This mechanism would lead to gradual reduction in length of the chromosomes and is fairly unlikely also for other reasons. Another. so far purely theoretic possibility. should be mentioned: The heritable constituent of the dms may be of viral origin rather than of chromosomal. Like most of the dms-carrying experimental tumors the SEWA has viral etiology; it was induced originally by polyoma virus and still carries the polyoma-
90
Heredirus 83 (1976)
G . LEVAN ET AL.
specific transplantation antigen (KLEINet a[. 1971; KLEINunpubl.). It has been shown recently that chromatin-like structures, “minichromosomes”, have been formed both in vivo and in vitro by SV40 and polyoma DNA after association with histones (GRIFFITH 1975; GERMOND et a]. 1975; CREMISI et al. 1976). The possible origin of dms from viral DNA has some attractive points, and experiments are being planned to test this possibility.
LATT,S. A . 1973. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. - Proc. Nur. Acud. Sci. 7 0 : 3385 3399 LEVAN. G. and LEVAN. A. 1975. Specific chromosome changes in malignancy: Studies in rat sarcomas induced by two polycyclic hydrocarbons. Hereditas 7Y: 161 . - 198 MANDAHL. N. and FREDC~A. K. 1975. Q-, G-, and C-band patterns of the mink chromosomes. - Heredirus 81: 21 1-220 MARK.J . 1967. Double-minutes. A chromosomal aberration in Rous sarcomas in mice. - Hereditus 5 7 : I -22 MARTINEAU. M. 1966. A similar marker chromosome in testicular tumours. Luncet ( I ) : 839% 842 F.. LEVAN. G . and MARK,J. 1972. The origin of MITELMAN, Acro Parhol. Micro-double-minutes in Rous rat-sarcoma. h i . Scand. A CIO: 428-429 NIELS~N K., 1976. A near-hexaploid Ehrlich-Lettre mouse ascites tumor line with low sensitivity to colchicine. HeriditusXJ: 105- 122 NORDENSKJBLD, B. 1964. Large scale production of polyoma virus in mouse ascites tumor cells in vivo. - Virology 24: 225- 227 C. and LINMAN, J. 1971. MicrochroPIERRE. R.. HOAGLAND. Cancer mosomes in human preleukemia and leukemia. 27: 160- 175 ROHME.D. 1975. Evidence suggesting chromosome continuity during the S-phase of Indian muntjac cells. - Heredirus 80: 145 149 SANDBERG, A. A,, SAKURAI, M. and HOLDSWORTH. R. 1972. Chromosomes and causation of human cancer and leukemia. VIII. DMSchromosomes in a neuroblastoma. - Cuncer 29: 1671-1679 J. S., WURSTER-HILL, D. H., PETTENGILL, 0. S. SHEPARD, and SORENStN. G. D. 1974. Giemsa-banded chromosomes of mouse myeloma in relationship to oncogenicity. Cytogiwet. Cell Genet. 13: 279-309 S ~ f f i ~H.t ~0.. , HELLSTRAM, I . and KLEIN.G. 1961. Transplantation of polyoma virus induced tumors in mice. C m w r Res. 2 i : 329 -337 M. M. ~ ~ ~ C L A R C.KM.E 1962. . SPRIGGS. A. 1.. BODDINGTON. Chromosomes of human cancer cells. Brrr. Med. J . ( 2 ) : 1431 -1435 SUMNER, A . T. 1972. A simple technique for demonstrating . Re.7. 75: 304-306 centromeric heterochromatin. - E . Y ~Cell WIENER,F. 1974. Studies on the chromosomal control of malignant behaviour by cell hybridization. - Thesis. Univ. Stockholm -
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Acknouledgments. - 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 BAYREUTHER, K. 1952. Der Chromosomenbestand des EhrlichAscites-Tumors der Maus. Z . Nururforsch. 7 h: 544-557 CREMISI, C., PIGNATTI. P. F., CROISSANT. 0. and YANIV.M. 1976. Chromatin-like structures in polyoma virus and simian J . Virol. 17: 204 -21 I virus 40 lytic cycle. FENYO.E. M.. WIENER.F.. KLEIN,G. and HARRIS,H. 1973. Selection of tumor-host cell hybrids from polyoma virusJ . Nut. (’onand methylcholanthrene-induced sarcomas. cer Insr. 5I: 1865-1875 J . E., HIRT. B., OUDET,P., GROSS-BELLARD. M. GERMOND, and CHAMBON. P. 1975. Folding of the DNA double helix in chromatin-like structures from simian virus 40. - Proc. Nar. Ai,ad. Scr. 72: lX43--1847 GRIFFITH,J. D. 1975. Chromatin structure: Deduced from a minichromosome. - Science 187: 1202- 1203 HAUSCHKA, T. S. and LEVAN, A. 1958. Cytologic and functional characterization of single cell clones isolated from the J . Nut. (’uncer Insr. Krebs-2 and Ehrlich ascites tumors. 21: 77-135 HENEEN, W. K 1976. HeLa cells and their possible contamination of other cell lines: Karyotype studies. - Hereditus X2: 217-248 U . , WIFNER,F. and HARRIS,H. 1971. KLEIN,G.. BRFGULA, The analysis of malignancy by cell fusion. I . Hybrids between J . Cell Sci. 8 : tumour cells and L cell derivatives. 659-672 ~~
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