Double Minute Chromosomes and a Homogeneously Staining Chromosome Region in C3H10T1/2 Murine Cells Transformed "In Vitro" by Proton Radiation Enrica Privitera, Giuliana Mosna, Elena Sala, Ivana Spiga, Fabio Gambaro, and Achille Ghidoni

ABSTRACT: F o u r foci (type II or type III) of transformed cells, isolated from the murine line C3HIOT1/ 2 after e x p o s u r e to p r o t o n radiations, were expanded and cytogenetically examined. While the overall numerical chromosome distributions were similar, there were some differences between the various cell lines with regard to the presence a n d frequency of specific-marker c h r o m o s o m e s

and to the colony-forming efficiency in soft-agarose medium. No association between any of these markers and the transformed phenotype could be established. However, in the line F4, derived from a type Il focus, n u m e r o u s double-minute chromosomes (DM) were observed after passage 22, and the phenomenon became more pronounced in the subclone C2. The finding of DMs in radiation-transformed cells is u n u s u a l . The DMs were observed in long-term subcultures, and in one of them they were partially replaced by a homogeneously staining chromosome region (HSR). DNAs from transformed cells of the line F4 a n d subclone C2 was digested with restriction enzymes and analyzed by Southern blotting with probes for seven oncogenes commonly amplified in cancer cells (c-myc, N-myc, N-ras, Ki-ras, Ha-ras, c-myb, c-abl) a n d with probes for the m o u s e MHC class I region. None of the regions tested was structurally altered or amplified in these transformed cells. The origin of the genetic material carried by DMs or homogeneously staining intrachromosomal regions (HSR) in cells of the line F4 and subclone C2, where it is believed to provide a selective advantage for in vitro growth, remains u n k n o w n .

INTRODUCTION The cytologic and molecular study of structural chromosome rearrangements found in many human and murine neoplasms has contributed to the understanding of the different mechanisms of neoplastic transformation [1-5]. Other types of changes in the genetic material, such as amplification of oncogene sequences that were found in some tumors [6, 7], have contributed to knowledge of important aspects of malignancy, such as tumor progression [8-11]. Amplified deoxyribonucleic acid (DNA) sequences have been cytogenetically described as supernumerary extrachromosomal elements called double-minute chromosomes (DM) or as homogeneously staining (HSR) intrachromosomal regions [12]. From the Dipartimento di Genetica e di Biologia dei Microrganismi dell'Universit~ di Milano, Via Celoria 26, 20133 Milano, Italy.

Address reprint requests to: A. Ghidoni, Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, 20133 Milano, Italy. Received A u g u s t 8, 1989; accepted September 26, 1989.

75 ~) 1990 Elsevier Science Publishing Co.. Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 49:75-86 (1990) 0165-4608/90/$03.50

76

E. Privitera et al. Structures of these types have been reported in many human tumors and tumorderived cell lines [6-11, 30], but only rarely in mammalian cells after transformation in vitro: in a study with spontaneously transformed NIH 3T3 mouse cells with DMs [13], two overexpressed distinct sequences associated with DMs (originated from mouse chromosome 10, not homologous to known oncogenes) have been isolated, but their role in transformed cells was not identified; DMs of unknown origin were also reported in another study with a spontaneously transformed murine cell line [14[. The present work regards the unusual finding of DMs in one of four mouse cell line C3H10T1/2 derivatives developed from type II or type III foci following irradiation with protons.

MATERIALS AND METHODS

Cell Culture The murine embryonic cell line C3H10T1/2 clone 8 [15] and four independently transformed clones derived from it were obtained from the Cell Biology Unit, Department of Physics, University of Milan, directed by Prof. L. Tallone. The cell lines were grown in standard conditions in a humidified incubator with 5% CO 2 at 37°C in Ham's F10 medium supplemented with 10% fetal calf serum (Flow), 100 IU/ml penicillin (Farmitalia) and 100 ~g/ml streptomycin (Squibb). Before, during, and after the proton-radiation treatments, until foci were isolated from a monolayer of untransformed cells, the cultures were maintained in Eagle's basal medium, in place of Ham's F10 medium, the other conditions being unchanged. The detailed conditions of culture and the irradiation procedure used to obtain morphologically transformed type II and type III foci, as well as their isolation, were reported elsewhere [16].

Colony-forming Efficiency in Agarose Cells from the original cell line C3H10T1/2 clone 8 and from each focus were tested for ability to grow in semisolid medium and to give rise to healthy colonies. The cells were seeded in medium Ham's F10 containing .33% soft agarose at an average density of 10 s cells/50 mm Petri dish and incubated at 37° for 3 weeks, as described by Lloyd [17]. Colonies with more than 100 cells were counted for evaluation of colony-forming efficiency or cloning efficiency CE (% of seeded viable cells capable of developing into a colony).

Cytogenetic Analysis Chromosome preparations were made by the standard air-drying technique. The chromosomes were then stained either with a buffered (pH 6.8) 4% Giemsa solution for chromosome counting or following sequential GTG [16] and CBG [19] banding methods. At least 20 well-spread metaphases for each cell clone were photographed through a Leitz Ortholux microscope and used for karyotype reconstruction.

Molecular Analysis Total DNA was extracted from cells of clone F4, the subclone C2 (with DMs), and the untransformed C3HlOT1/2 clone 8 cells, and Southern blots were prepared according to standard protocols [20]. The filters with DNA fragments obtained by digestion with Eco RI or Hind III were hybridized in 50% formamide, 5 × SSC, 5 × Denhardt's

77

DMs in Radiation-transformed Murine Cells

Table 1

Chromosome n u m b e r distributions and colony-forming efficiency of mouse untransformed C3H10T1/2 cells and four proton-radiation transformed cell lines derived from type II and type III foci

Line

Character°

Passage

C3HlOT1/2 F1 F2 F3 F4

U T (Ill} T (II) T (III} T (II)

20 29 20 22 23

Modal chromosome No. FWHM~ 74 71 74 73 74

+- 5 -+ 4 -+ 4 -+ 2 -+ 6

Colony forming efficiency % (CE} + Sc 0.15 4.0 2.4 0.6 11.0

+- 0.08 -+ 2.0 -+ 1.0 -+ 0.3 -+ 5.0

o u, untransformed; T, transformed (from type II or type llI loci). b FWHM,full width half maximum assuming Gaussian distribution. Mean values from three experiments; S, standard deviation from the mean or standard error, whichever greater.

solution, .1% SDS and 100/~g/ml sonicated salmon sperm DNA at 42°C for 20 hours. Probe DNA fragments were labeled with 32p by the r a n d o m priming m e t h o d [21]. Filters were washed u n d e r either stringent {.2 x SSC at 65°C} or m o d e r a t e l y stringent (1 x SSC at 65°C} conditions. The following DNA probes were used: Oncogenes: c-myc [22], N-myc [23], N-ras [24], Ki-ras [25], Ha-ras [25], c-myb [26] and c-abl [27]. Mouse H-2 class I region: MHC 1 [28] and pH-2III [29]. RESULTS Four cell lines derived from type II or type III foci, designated as F1, F2, F3, and F4, respectively, were cytogenetically analyzed. As shown in Table 1, only moderate variations in the n u m e r i c a l c h r o m o s o m e distributions were observed, w i t h no significant difference b e t w e e n the four transformed and the untransformed cell lines. On the contrary, differences in the cloning efficiency in soft agarose were found, with values of 4.0, 2.4, .6, and 11%, respectively, for lines derived from the four loci {between the 20th and the 33th passage}, and a value of .15% for the untransformed C3H10T1/2 (at the 20th passage}. Cells derived from morphologically transformed type II or t y p e III foci are k n o w n to induce tumors with high frequency w h e n injected in syngenic mice [30]. The s t u d y of several reconstructed karyotypes showed a remarkable variability in the n u m b e r and aspect of marker chromosomes {Fig. 1}. A n interesting observation was m a d e in line F4: n u m e r o u s s u p e r n u m e r a r y double minute (DM} chromosomes a p p e a r e d after the 20th passage {Table 2}. The percentage of cells with DMs was initially low but r a p i d l y increased with successive passages {Fig. 2}, and the n u m e r i c a l presence of DMs became also more consistent, as s h o w n in Figure 3. A subclone of line F4, n a m e d C2, was isolated after plating cells in m e d i u m w i t h soft agarose. This clone was characterized by r a p i d growth and by 8 5 % - 9 0 % of cells w i t h n u m e r o u s DMs. In another subculture of line F4, a homogeneously staining region (HSR} was noticed in a marker c h r o m o s o m e that was identified as a rearranged t(12;X}, s h o w n in Figure 4. In this subculture, the percentage of cells with DMs was observed to

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E. Privitera et al.

Harkt,r chromosomes

Original chromosomes

Presence of markers +

i:

J t (11 q; 5 q)

11

Cl8, FI, F3, F4

F2

5

!

F2, F3

Cl8, FI. F4

iso9 q

iso 16 q

Cl8, FI, F2, F4

F3

Cl8. FI, F2, F4

F3

16

~Q lq÷

M5

1

i

4q-

M6 2q-

I

F2

cz8. F,, F3. F4

4

m

! 2

Figure 1 Identification and distribution of six marker chromosomes in the untreated C3H10T1/2 clone 8 (C1 8) and in four transformed clones (F1, F2, F3, and F4).

decrease, together with a proportional increase of cells with a HSR region (Fig. 5). DMs or HSRs were never observed in untransformed IOT1/2 cells. Another interesting observation was m a d e in cells of the line F4: some of the metaphases (1%-10% in different subcultures) exhibited, in association with a high n u m b e r of DMs, a remarkable c h r o m o s o m e instability, with numerous chromosome fragments, c o n d e n s e d single chromatids and ring chromosomes (Fig. 6). It is worth mentioning that the F4 cells had the highest CE value (11%) in sofl-agarose medium. In an attempt to identify the origin and nature of the p r e s u m a b l y amplified DNA

79

DMs in Radiation-transformed Murine Cells

100-

Passage 24 th

0-

211th

50-

38th

50-

,4,

47 th

01-20

4140 IH00 ~ >100

No o~ DMs/cell

Figure 2 Relative frequency of cells with different numbers of double minutes in various passages of the transformed line F4.

sequences present in the form of DMs or HSR regions, F4 cells were analyzed by Southern blotting. The total DNA extracted from F4, the subclone C2, and the untransformed C3H10T1/2 cells, after digestion with Eco RI and H i n d III restriction enzymes, was h y b r i d i z e d with radioactive probes for seven of the oncogenes that are more c o m m o n l y amplified in some tumor cells or in cell lines derived from them, such as c-myc, N-myc, N-ras, Ki-ras, Ha-ras, c-myb, c-abl. The hybridization intensity was found to be similar in all cases, indicating that none of the seven oncogenes tested was amplified in the F4 cells and its subclone C2. The Southern blotting analysis was

80

E. P r i v i t e r a et al.

O f

Figure 3

"

i

Numerous double minute chromosomes (about 80) in a cell of suhclone C2 of the

transformed line F4.

Table 2

R e l a t i v e v a r i a t i o n of t h e n u m b e r of D M s p e r cell w i t h p a s s a g e s i n t h e l i n e F4 (data f r o m 100 o b s e r v a t i o n s for e a c h passage) % of cells at passages

No. of DMs/cell

24

28

38

47

0 1-20 21-40 41-80 81-100 Over 1O0

80 13 4 2 0 1

23 50 15 5 O 5

34 32 19 5 2 5

25 37 24 7 3 11

DMs in Radiation-transformed Murine Cells

81

l/ ! tO o O

# ~Q

~b

!

! f

Figure 4 A G-banded metaphase of the transformed line F4 with a HSR in marker chromosome (indicated by the arrow).

e x t e n d e d by use of a probe for the mouse MHC, H-2 region, since alterations of HLA class I genes have been found [31] in a significant proportion of h u m a n colon cancers (2 cases of rearrangement and I case of amplification in a sample of 12). In this study w i t h two probes (MHC 1 and pH-2III) for class I antigens, no rearrangement or amplification in the mouse H-2 region was observed.

DISCUSSION The n u m e r i c a l c h r o m o s o m e distribution in radiation-transformed cell lines and in the untreated cell line C3H10T1/2 did not significantly differ. It was impossible to establish that any of the n u m e r o u s marker chromosomes observed in both the radiation transformed cell lines and in the untreated 10T1/2 cells had a role in transformation.

82

E. Privitera et al.

~. 0 ...... ......

---.,

W



,/" •

.°°° ~°°

~)

1

I

oM IDl

o°

o,

I

30

|

I

50

I

!

70

Figure 5 Relative frequency of cells with DMs (solid circles), HSR (solid triangles), and DMs or HSR (square symbols) in a subculture of the transformed line F4 (100 cells counted for each point).

A high degree of chromosomal instability of the kind described in F4 cells has also been observed by other researchers in a subline of untreated HeLa cells with n u m e r o u s DMs [32], in a study of mouse l y m p h o m a cells after treatment with h y d r o x y u r e a [33], and in mouse C3H10T1/2 cells selected for resistance to methotrexate, exhibiting n u m e r o u s DMs ( u n p u b l i s h e d observations made in our laboratory). Similar findings have been described in vivo in two unrelated h u m a n pathologies [34]. It was suggested that the generation of DMs is favored by an i n d u c e d genomic instability [33]; alternatively, one could speculate that a high number of DMs, w h i c h have been shown to replicate early in the S phase [36], could interfere with the cells' replication processes and contribute to chromosomal instability. It is not known, however, whether the c h r o m o s o m a l instability observed in such different materials and conditions has a c o m m o n origin and/or a similar biologic significance. The most significant result of the present study is the finding of DMs and HSR material in one out of four cell lines derived from foci after irradiation with protons. DMs and HSR are generally regarded as cytologic evidence of amplified DNA sequences [12]. This may be regarded as a very u n u s u a l finding in radiation-transformed cells. It should be noted, however, that only a few cell types have been used in transformation experiments with radiations, and that no systematic cytologic or molecular investigation of such transformed cells has been carried out. The generation of DMs in some h u m a n tumors and during in vivo growth of e x p e r i m e n t a l l y i n d u c e d tumors has been thought to reflect changes in the copy n u m b e r of genes involved in malignancy such as oncogenes [1, 5], genes of the HLA region [31], satellite DNA or ribosomal RNA genes [35]; the meaning of other overexpressed DNA sequences, as in the case of s p o n t a n e o u s l y transformed mouse 3T3 cells [14] with DMs, was not established. In our study, the persistence of DMs in F4 cells and in the subclone C2, and the partial replacement of DMs by a HSR in long-term subcultures, are in agreement with similar observations by other workers [36], and with their suggestion that these altered c h r o m o s o m e structures may provide some advantage to cell growth. In different investigations, double minutes have been shown to anticipate the formation

83

-

I

.

o

Figure 6 A metaphase of the transformed line F4 with numerous double or single minutes associated with various chromosome anomalies such as ring chromosomes, single chromatid chromosomes, and chromosome fragments.

of HSR regions [36, 37, and others, including the present report], or to follow the breakdown of a HSR [38, 39]. In many cases, the presence of amplified genetic material has been associated with the more advanced steps of neoplastic transformation [8-11]; in others, the meaning of the presence of DMs in untreated HeLa cells [32] or in in vitro transformed murine cells [13,14], remained unknown, but some authors suggested that the amplified genome could represent sequences involved in basic cellular functions playing a role in the maintenance of the transformed phenotype [14, 40]. We were unable to identify the presumably amplified genetic material present in DMs of F4 cells. We can only exclude the involvement of a little portion explored of the mouse genome (seven oncogenes and the MHC class I region) in the production of the cytologically abnormal structures observed. These are now being investigated with other methods available for the identification of amplified sequences of unknown origin [41]. The molecular characterization of sequences contained in DMs and in the HSR that we have described in F4 cells will be useful for the characterization of

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genetic c h a n g e s o c c u r r i n g in o t h e r r a d i a t i o n - t r a n s f o r m e d cells [42] and in the s t u d y of the e v o l u t i o n of m a l i g n a n c y .

This investigation was supported by grants of the MPI 40% and with a contribution from the CEC contract BI6/O0177/I(s). The untreated cell line C3H10T12 cl 8, and the four proton-radiation transformed-cell foci were a generous gift of Prof. L. Tallone (Director of the Cell Biology Unit, Dept. of Physics) who obtained the transformed C3HIOT1/2 ceils and isolated the loci together with Prof. D. Bettega, Dr. P. Calzolari, and Dr. A. Ottolenghi. The nucleic acid probes were kindly supplied by Dr. M. Pierotti and Dr. G. Parmiani of the Istituto Nazionale per lo Studio e la Cura dei Tumori (Milano). The authors wish to thank Prof. S. Ottolenghi and Dr. R. Taramelli (Dipartimento di Genetica e di Biologia dei Microrganismi, Milano) for assisting the molecular investigation.

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