Specific Chromosomal Aberrations Correlated to Transformation in Chinese Hamster Cells Silvana Simi, Antonio Musio, Lucia Vatteroni, Antonio Piras, and Giuseppe Rainaldi
Cytogenetic changes were investigated during the spontaneous progression of CHEF18 Chinese hamster cells towards tumorigenicity. We farther report the chromosomal characterization of a series of spontaneous anchorage-independent clones, as well as of a series of tumor-derived cell lines resulting from injection of late passage cells in nude mice. The high karyotypic homogeneity (presence of four marker chromosomes strictly associated in all the metaphases analyzed) in all clones and tumorderived cell lines prompted us to alter this specific pattern of chromosomal aberrations in order to identify which if any of the aberrations were more strictly related to transformation. For this purpose we treated a tumor-derived cell line with Colcemid and analyzed the reversion of anchorage-independent phenotype in the subclones showing an altered association of the four m a r k e r chromosomes. We conclude that two of four marker chromosomes contribute to anchorage independence. ABSTRACT:
INTRODUCTION Most h u m a n [1, 2] and animal tumors [3, 4] are characterized by c h r o m o s m n a l aberrations. The findings of consistent c h r o m o s o m a l changes in m a n y hematopoietic malignancies [5] indicate that specific aberrations are involved in neoplastic transformation [6, 7]. However, the relationship between most of these aberrations and specific p h e n o t y p i c changes is still unclear, making it difficult to u n d e r s t a n d if they are causative or secondary events occurring during the neoplastic process. A n a p p r o a c h to investigate this problem is to examine cells at different steps of in vitro propagation, to determine if specific patterns of c h r o m o s o m a l changes can be observed and correlated to the expression of the transformed phenotype. CHEF 18, an immortalized, non-tumorigenic, fibroblastic Chinese hamster cell line [8], has been extensively used for genetic analysis of tumorigenesis [9, 10]. We have recently found that after prolonged serial passage this cell line undergoes s p o n t a n e o u s progression to tumorigenicity. This process is a c c o m p a n i e d by a r e d u c t i o n of proliferative heterogeneity [11] and increased ability both to grout in soft agar and to undergo gene amplification [12]. In this paper we report the karyotypic changes of CHEF18 cells during this progression and the chromosomal
From the Genetica e Biochimica Tossicologica, Istituto di Mutagenesi e Differenziamento CNR, Pisa, Italy. Address reprint requests to: Dr. Silvana Simi, IMD, Via Svezia 10, 56124, Pisa, Italy. Received February 24, 1992; accepted May 8, 1992. © 1992 Elsevier Science Publishing Co., Inc.
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characterization of a series of clones specifically selected at different stages of progression. All the selected clones showed only one specific and recurrent pattern of chromosomal changes c o m p r i s e d of four marker chromosomes. To determine the stringency of the four markers to the transformed p h e n o t y p e , we tried to obtain clones with different dosages of the four marker chromosomes. We have p r e v i o u s l y s h o w n that Colcemid, a microtubule disrupting agent k n o w n to i n d u c e p o l i p l o i d y [13] and chromosomal non disjunction [14], was also able to induce altered segregations [15] leading to n u m e r i c a l l y d i p l o i d cells, whose karyotypes presented a loss-gain of one chromosome and gain-loss of another [161. Thus we treated a t u m o r - d e r i v e d cell line with Colcemid and here report the c h r o m o s o m a l analysis of i n d u c e d subclones. Subclones were selected on the basis of altered segregations of the four marker c h r o m o s o m e s and challenged in soft agar. The results show that two markers cosegregated with the loss of anchorage i n d e p e n d e n c e . MATERIALS AND METHODS Cell Line
CHEF18 is an essentially d i p l o i d , untransformed, and nontumorigenic Chinese hamster cell line [8, 17]. This line was obtained by Prof. R. Sager (Dana-Farber Cancer Institute, Boston, MA) at passage 14. A standard protocol for propagation was used [11]. Briefly, the cells were c u l t u r e d in Dulbecco's Modified Eagle's m e d i u m s u p p l e m e n t e d with 10% fetal calf serum and in an a t m o s p h e r e of 6% CO2. Every 3 days the cells were t r y p s i n i z e d and a s a m p l e of 5 x 105 cells was propagated. With this schedule, an average ot four cell doublings occurred between two trypsinizations. 81 Cancer Genet Cytogenet 62:81 87 (1992) 0165-4608/92/$05.00
82
Cytogenetic Analysis E x p o n e n t i a l l y growing cells were treated with Colcemid, harvested, incubated with KC1 0.075 M, and fixed in methanol: acetic acid 3:1, as previonsly described [18]. Chromosome preparations were G-banded according to a trypsin digestion procedure [191. The proposed nomenclature of Ray and M o h a n d a s for Chinese hamster chromosomes was used [20].
Statistics A reliable statistical analysis using a basic program developed in our laboratory [211 was used to distinguish nonr a n d o m events from the casual a c c u m u l a t i o n of aberrations that can occur as a consequence of random distribution.
Soft-Agar Assay A n c h o r a g e - i n d e p e n d e n c e test was carried out as described elsewhere [12]. A p p r o p r i a t e concentrations of cells were cultured in semisolid agar m e d i u m and colonies were stained with cristal violet 3 weeks later. Only colonies equal or greater than 250 /am in diameter were counted. I n d i v i d u a l clones were isolated from soft-agar and chromosomal analysis was performed 6 - 1 0 days after isolation.
Tumorigenicity Assay Cells (8 × 106) were injected s u b c u t a n e o u s l y into 17 athymic Balb/C male and female mice. Tumors bigger than 5 m m in d i a m e t e r were scored as positive. Cell lines were established from eight i n d e p e n d e n t tumors and k a r y o t y p e d 6 - 1 0 days after isolation.
Colcemid Treatment Cultures were set up the day before the treatment at a density of 8 x 105 cells per 60-ram Petri dish. Colcemid (Boehringer, 0.025/xg/ml) was a d d e d for 16 hours. Following treatment, cells were w a s h e d and reincubated in fresh m e d i u m for a further 24 hours. Cells were then t r y p s i n i z e d and plated into 100-ram Petri dishes at 50,100, and 200 cells per dish. At 2 weeks, i n d i v i d u a l colonies were isolated using glass c y l i n d e r s coated with silicone lubricant and e x p a n d e d for c h r o m o s o m a l analysis.
RESULTS Karyotype Evolution We have a n a l y z e d the karyotypic evolution of CHEF18 cell line as it s p o n t a n e o u s l y became tumorigenic. The analysis of cells at different passages (Table 1) showed that in the early phases of in vitro propagation different karyotypes were generated and cells with an extra copy of the long arm of c h r o m o s o m e 3 (3q) and with a marker c h r o m o s o m e originating from c h r o m o s o m e 4 were significantly in excess. The structural alteration involving c h r o m o s o m e 4 gave rise to a rearranged c h r o m o s o m e 4 q + with a larger than normal long arm owing to insertion of an u n k n o w n segment distal to band 4q25. From the m i d d l e phase of propagation (after 60 days in culture), while the extra 3q is lost and 4 q + retained, a steady increase of karyotype instability occurred, which
S. Simi et al. finally resulted in the significant presence of other altered chromosomes originating essentially from c h r o m o s o m e s 7, 8, and 9. The structural alterations involving c h r o m o s o m e 7 consisted both of a c o m p l e t e deletion of the short arm generating a c h r o m o s o m e formed by the long arm of chromosome 7 (7q) and of the formation of a c h r o m o s o m e 7 p + : this may have occurred by centromeric fusion between 7q and another c h r o m o s o m a l element or by juxtaposition of a c h r o m o s o m a l segment (possibly from 8p, which is contemp o r a n e o u s l y deleted) on the short arm of 7. Because of the observation that when a 7q was present the 7 p + was not, and vice versa, and of the presence of a fragile site (18) at the centromere of c h r o m o s o m e 7, it seems likely that the marker 7p + represents a translocation of the whole short arm rather than a juxtaposition. However, the origin of chromosmnal material in 7p ÷ could not be determined, as the inserted material consisted of a single band. making identification impossible. Alterations involving c h r o m o s o m e 8 consisted of both c h r o m o s o m e 8q, with a c o m p l e t e l y deleted short arm, and, much more frequently, marker 8 p - , having a small deletion of the distal part of short arm at band 8p16. Finally, the structural alteration of c h r o m o s o m e 9 consisted of a marker 9q + , w h i c h seems to have a differentially staining amplified region, often of variable length, in the long arm. A metaphase presenting 4 q + , 7 p + , 8 p - . and 9q + marker c h r o m o s o m e s is s h o w n in Figure 1. The p o p u l a t i o n tended to r e m a i n d i p l o i d in all stages of progression, although a small fraction of s p o r a d i c a l l y occurring a n e u p l o i d cells was also present (data not shown).
Analysis of Anchorage-Independent Clones and Tumor.Derived Cell Lines A n c h o r a g e - i n d e p e n d e n t (AI) clones derived from m i d d l e passage cells seeded in soft agar and t u m o r - d e r i v e d cell lines resulting from injection of late passage cells in n u d e mice were cytogenetically a n a l y z e d to d e t e r m i n e if specific chromosomal changes correlated to both in vitro and in vivo transformation parameters. Tables 2 and 3, respectively, show the c h r o m o s o m a l changes found in five AI clones and in eight t u m o r - d e r i v e d cell lines. All clones and cell lines d i s p l a y e d a unique karyotypic pattern characterized by the simultaneous presence of four marker chromosomes, 4q + , 7p + , 8 p - , and 9q + , w h i c h were also observed, at various frequencies, during in vitro propagation.
Analysis of Clones and Suhclones Induced by Colcemid in 835T2 Tumor-Derived Cell Line To obtain clones with different dosage of marker chromosomes, 835T2 t u m o r - d e r i v e d cell line was treated with Colcemid. The G-banding cytogenetic analysis of 65 Colcemidi n d u c e d clones shows that eight clones d i s p l a y e d altered c h r o m o s o m e segregations, with different c h r o m o s o m e s r a n d o m l y involved in a n o m a l o u s segregations {Table 4). Of particular interest were clones colc 3/8 and colc 3/ 15, both presenting a deleted c h r o m o s o m e 7q originating by a centromeric break, one from marker c h r o m o s o m e 7p + {clone colc 3/8) and one from normal c h r o m o s o m e 7 (clone
Specific Chromosomal Aberrations and Transformation Table 1
83
Chromosome markers found at different intervals of in vitro propagation I n t e r v a l (in days)
Chromosome marker lp + lq lq 2p2q3p 3q 4q 4q+ 5q 5q÷ 6q 6q6q-
Band involved lp22 1ten lq22 2p28 2q31 3p16 3ten 4ten 4q25 5cen 5q23 6cen 6q16 6q24
7q } 7ten 7p+ 7q7q23 8p 8p16 8q 8cen 9q+ 9q22 Xq Xcen XqXq18 Yq Ycen Total m e t a p h a s e s analyzed
3
6
15
27
42
54
60
78
93
2
99
1
2
126
180
3
2
3
1 2
1 1
1 2
1 5 **~' 1 1
4** 10"**
1 4**
9**
6**
2
6***
8*** 1
2 1
2
1 4** 7***
1 1 2 14"**
1 1
36***
30***
1 2
2
3
1
1
1 2
1
1
4**
1
5***
1
2
1
5
1
1
2
45
40
40
40
1
2
36*** 1 2
4 15"**
4 24***
10"** 24***
7*** 3 5*
24*** 4 15"**
19"** 3 14"**
10"** 2 1 1
1 50
2 1
1
1
32***
40
6** 1 40
2
40
40
40
Cell s h o w i n g the p r e s e n c e of the four m a r k e r c h r o m o s o m e s .
~ ~i i¸¸~¸~¸
31"**
4
16"** 4 4 6*** 4 1 1
4 33***
2
21"** 13"** 12"** 2 14"**
2
" Asterisks indicate bands with a significant excess of lesions (*:p < 0.05: ** :P < 0,01; *** :p < 0.001).
Figure 1
105
q÷
7
t~
P÷
dsp_
40
40
40
84
Table 2
S. Simi et al.
Marker chromosomes found in five spontaneous a n c h o r a g e - i n d e p e n d e n t clones Marker t:hrolIlosome
Clone ~ 677/1 677/2 677:3 677:4 677/5
Metaphases analyzed
4q +
7p +
15 10 15 10 10
12 10 11 9 9
15 8 13 10 10
8p
9q
15 10 15 9 10
14 10 15 10 10
DISCUSSION
" All c l o n e s s h o w e d a d i p l o i d m o d a l c h r o m o s o l n e n u m b e r .
colc 3/15). Clone cole 1/6 is also interesting; it is aneuploid, having lost the normal copy of chromosome 7. To further disrupt the association of the four markers and to collect other clones with altered segregations, clone colc 3/8 was treated with Colcemid. The analysis of 28 Colcemid-induced subclones {see Table 5) identified four subclones displaying altered segregations. It is worth noting clone colc 4/23 lacking marker chromosome 8 p - and clone colc 4/69 lacking marker chromosome 9q +.
Anchorage-Independence Analysis in Clones and Subclones with Altered Segregations in Marker Chromosomes Table 6 shows the soft agar (SA) colony-forming ability of parental 835T2 tumor-derived cell line and of clones and subclones with altered segregation in the marker chromosonles.
While two of five clones were similar to the tumorderived cell line, three clones grew in soft agar with low efficiency. Table 7 shows the relative plating efficiency (RPE, the ratio of plating efficiency in soft agar to plating efficiency in liquid medium) correlated with the presence or absence of marker chromosomes. All chines and subclones retained the normal copy of chromosome 4 and the marker 4q +. When normal chromosome 7 is partially (clone colc 3/15) or completely lost (clone cole 1/16) growth in soft agar is not affected, whereas when the anomalous short arm of marker 7 p + is lost (clones colc 3/8 and 4/69) the ability to grow in soft agar is
Table 3
reduced. When, concurrently, marker 8 p - is also losl (clone cole 4/23) soft agar growth is greatly reduced. The presence or absence of marker 9 q + seemed u n i m p o r t a n t because two clones {cole 3/8 and 4/69), one with and the other lacking 9 q + , showed the same RPE. This analysis clearly shows that reversion from the anchorage-independent phenotype co-segregates with the loss of chromosomes 7p + and 8p - , whereas the presence or absence of chromosomes 4q + and 9q ~- did not affect soft agar growth.
In this stud), a karyotypic analysis of CHEF18 Chinese hamster cells during the spontaneous progression to tumorigenicity was carried out. CHEF18 Chinese hamster cells presented genetic instability during serial propagation; in addition to a broad spectrum of random chrmnosomal changes, there are n o n - r a n d o m alterations which are found to precede and then accompany the acquisition of tumorigenicity. In c o m m o n with other works [22, 23], an extra copy of the long arm of chromosome 3 {3q) (where two oncogenes have been mapped [24]), was the most frequent abnormality during the early steps of progression. Trisomy 3q, together with contemporary loss of the associated short arm (3p) (where the presence of a tumor suppressor gene has been suggested [25]), is supposed to be sufficient to induce tumorigenicity in Chinese hamster cells [22]. Our data suggest that cells with trisomy 3q had a selective advantage during the early phase of in vitro progression. Successively, n u m e r o u s other heteroploid karyotypes were generated, not necessarily all related to transformation, probably reflecting somatic events of the ongoing tumorigenic progression. Other chromosomes involved in the formation of markers were 4, 7, 8, and 9. One of the markers, 4q +, occurred very early during progression: the other three appear to occur later. Changes involving these chromosomes have been observed during transformation in Chinese hamster system; an extra copy of chromosome 4q was found during the early stages of liver carcinogenesis (26), whereas trisomy of chromosome 8, as well as the presence of 8q marker, were found during s p o n t a n e o u s neoplastic evolution of Chinese hamster cells 127].
Marker chromosomes found in eight tumor-derived cell lines Marker chromosome
(;ell line" ES1 ES2 835T1 835T2 835T3 835L4 834L5 834L6
Metaphases analyzed 10 10 6 11 10 9 11 13
2q -
3p -
3q
4q
2
1
3
1
" All cell lines showed a diploid modal chromosome number.
4q + 9 9 5 10 7 9 9 9
5q
6q --
1
1
7p t
8p -
9q +
9 10 6 11 10 9 11 13
9 10 6 11 10 9 12 1'3
1(1 10 5 11 9 9 i1 1'3
Xq
Yq
2 4 1 1 1
Specific C h r o m o s o m a l Aberrations and Transformation
Table 4
85
G-banding c h r o m o s o m a l analysis of clones i n d u c e d by Colcemid in 835T2 t u m o r d er i v ed cell line
A)" Clones with parental karyotype 51
Clones displaying altered segregations
Clones with hyper diploid or tetra ploid karyotype
Total clones analyzed
6
65
8
B)b Clone 835T2 (parental one) Colcl/5 Colc 1/6 Colcl/12 Colc 1/15 Colc 2/6 Colc 3/7 Colc 3/8 Colc 3/15
Modal chromosome number 22 25 21 22 (18-23) 24 22 22 22
Chromosomal constitution C -4+4q+,
-7+7p+,
-8+8p-,
-9+9q+,
-4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+,
-7+7p+, -7+7p+, -7+7p+, -7+7p+, -7+7p+, -7+7p+, -7p++7q, -7+7p+,
-8+8p-, -8+8p-, -8+8p , -8+8p-, -8+8p-, -8+8p-, -8+8p-, -8+8p-,
-9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+,
+Y
+5,+9q, -7 -6+6q -4+4q +5,+Y -9+9q -7+7q
° A) Chromosomal constitution of all clones. ~'B] Detailed chromosomal constitution of the eight clones displaying altered segregations. ': Chromosome alterations were recorded when more than two thirds of the metaphases examined presented the alteration. Never less than 15 metaphases were analyzed per each clone.
In the CHEF system, the long arm of c h r o m o s o m e 4 was found differently rearranged in cells transfected with a plasmid [28], whereas m o n o s o m y of c h r o m oso m es 7 and/or 8 is one of the most consistent aberrations in transformed cells [25]. C h r o m o s o m e 9 with amplified regions was also sporadically found during azacytidine-induced tumorigenesis [22]. The breakpoints that form two of the observed rearranged c h r o m o s o m e s (3q and 7p + ) were in regions w h er e fragile sites are expressed; 3 cen and 7 cen have been reported to be folate-sensitive fragile sites in Chinese hamster
Table 5
c h r o m o s o m e s [18]. These data further em p h asi ze that fragile sites are predisposing factors for c h r o m o s o m a l changes and can contribute to the creation of stable c h r o m o s o m e rearrangements. In order to study the p h e n o t y p i c c o n s e q u e n c e s of these ch r o m o so m al changes, AI clones and t u m o r - d e r i v e d cell lines were analyzed. The data s h o w e d a constant presence of four marker chromosomes, w h i c h are u n i f o r m l y retained in all selected clones and t u m o r - d e r i v e d cell lines, indicating that transformation can occur w i t h o u t extensive genetic instability. Other karyotypes may have been generated, but,
G-banding c h r o m o s o m a l analysis of subclones i n d u ced by Colcemid in clone colc 3/8
A)~ Subclones with parental karyotype
Subclones displaying altered segregations
Subclones with hyper diploid or tetraploid karyotype
Total subclones analyzed
23
4
1
28
B) ~
Clone Colc 3/8 (parental one/ Colc 4/13 Colc 4/23 Colc 4/69 Colc 4/88
Modal chromosome number 22 21 Ca)21 b)22 a)(20-23) b)(21-23) 22
Chromosomal constitution ° -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q,
°'~'see Table 4. ': Subpopulation a and b were present in approximately equal proportions.
7p+ +7q, -7p+ -7p+ -7p+ -7p+ -7p+ -7p+
+7q, +7q, +7q, +7q, +7q, +7q,
-8+8p-,
9+9q+
-8+8p-, -8p-, 8p , -8+8p-, -8+8p-, -8+8p-,
-9+9q+, -9+9q+ 9+9q+, -9+9q+ -9q+ -9+9q+,
-y +7
-X+Yq
86
Table 6
S. Simi et al.
Anchorage i n d e p e n d e m : e in 835T2 tumorderived cell line and in clones and subchmes displaying altered segregations of marker chromosomes
{2lone
P.E. in soft agar {%}
P.E. in DMEM (%}'
835T2 cole 3/15 col{: 1/6 col{: 3/8 cole 4/69 colc 4/23
51.2 41.9 37.5 7,4 7.3 1.1
100 100 100 61 60 49
" These results represent the average of two experimenls.
if so, they were lost, and one cannot e x c lu d e that further extensions of these studies could reveal other c h r o m o s o m e combinations that play a role in transformation and tumorigenicity. Different "scenarios" have been proposed for clonal evolution of neoplasms that can either evolve toward greater diversity (genetic divergence) or toward h o m o g e n ei t y (genetic convergence) [29]. U n d e r our standard culture conditions, the balance b e t w e e n genetic instability and selective pressure favored the emergence of a p r e d o m i n an t karyotype. To assess the functional role of the various c h r o mo so m al abnormalities found, C o l c e m i d - i n d u c e d clones with altered marker c h r o m o s o m e distribution were tested for their ability to grow in soft agar, because anchorage independence is generally regarded as the best tissue-culture correlate of tumorigenicity [30, 31]. The pattern of c h r o m o s o m e loss suggested that marker c h r o m o s o m e s 7p + and 8p - may be i n v o l v e d in transformation; clones w h i c h have lost either of these chromosomes grow poorly in soft agar, whereas the presence or the absence of 4q + and 9q + were without effect. It has been s h o w n [17, 32] that segregation of anchoragei n d e p e n d e n t subclones from somatic hybrids between anc h o r a g e - i n d e p e n d e n t and a n c h o r a g e - d e p e n d e n t CHEF cells was associated with the loss of all or part of one copy
Table 7
Relative plating efficiency (RPE] versus presence/ absence of marker c h r o m o s o m e s and their normal homologous Chromosome
Clone
RPE~'
835T2 Colc 3/15 Colc 1/6 Colc 3/8 Colc 4/69
51.2 41.9 37.5 12.1 12.1
Colc 4/23
2.2
~'see Results.
4 4, 4, 4, 4, 4, 4, 4, 4,
4q+ 4q+ 4q+ 4q+ 4q+ 4q+ 4q+ 4q+
7 7, 7p+ 7q, 7p+ 0, 7p+ 7, 7q 7, 7q 7, 7q 7, 7q 7, 7, 7q
8 8, 8, 8, 8, 8, 8, 8, 8,
8p8p8p8p8p8p0 O
9 9, 9, 9, 9, 9, 9, 9, 9,
9q+ 9q+ 9q+ 9q+ 0 9q+ 9q+ 9q+
of c h r o m o s o m e 1. In our work, however, we have found no significant alterations in c h r o m o s o m e 1. It appears that loss of genetic material from the short arm of c h r o m o s o m e 8 and loss or relocation of genetic material from the short arm of c h r o m o s o m e 7 may represent fundamental events in the spontaneous transformation of Chinese hamster cells, presumably conferring an altered growth requirement revealed as a n c h o r a g e - i n d e p e n d e n c e growth. The presence of specific c h r o m o s o m e aberrations is a signal that unidentified changes in the genome may have occurred. The observation that one alteration is a deletion and the other a translocation suggests that possible genetic changes could be specific allelic loss of suppressor genes and activation of protooncogenes. The fact that suppressor genes are supposed to be located on c h r o m o s o m e 7 and 8 [25] and that an oncogene, K-ras, has been m a p p e d on c h r o m o s o m e 8 [241, further supports the hypothesis. Tire other two genes m a p p e d on c h r o m o s o m e 8 are GAPD arrd TPY1, w h i c h are linked with K-ras. It is perhaps noteworthy that distal and terminal deletions of h o m o l o g o u s human 12p containing the same three genes are reported from various h u m a n n eo p l asm s [33]. The identification of other genes on c h r o m o s o m e 7 and 8 might help in understanding the relationship between c h r o m o s o m e aberrations, gene expression, and tumorigenic process in Chinese hamster sy'stem.
This work was partially supported by AIRC (Italian Association for Cancer Research) and CNR (National Research Council) Target Project "Applicazioni Cliniche della Ricerca Oncologica". Lucia Vatteroni is the recipient of a fellowship from AIRC. The authors thank Dr. E. Giulotto and Dr. M. Minks for critical review of the manuscript, and Mrs. G. Cecchi for typing.
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XXI. Suppressor genes in CHEF cells. Somat Cell Mol Genet 11:25-34. Rainaldi G, Pinto B, Piras A, Simi S, Citti L (1991): Reduction of proliferative heterogeneity of CHEF18 Chinese hamster cell line during the progression toward tumorigenicity. In Vitro Cell Dev Biol 27:949-952. Vatteroni L, Piras A, Mariani T, Caligo MA, Rainaldi G: Concurrent emergence of anchorage-independence and gene amplification during the in vitro propagation of CHEF18 Chinese hamster cells. Cell Proliferation (submitted) Harris M (1971): Polyploid series of mammalian cells. Exp Cell Res 66:329-366. Cox M (1973): A quantitative analysis of Colcemid-induced chromosomal non disjunction in Chinese hamster cells in vitro. Cytogenet Cell Genet 12:165-174. Rainaldi G, Flori L, Colella CM, Mariani T, Piras A, Simi S, Simili M (1987): Analysis by BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture. Mutation Res 177:255-260. Simi S, Colella CM, Mariani T, Piras A, Rainaldi G (1988): Qualitative analysis of chromosomal evolution in a Colcemidtreated Chinese hamster population. Teratogenesis Carcinog Mutagen 8:45-54. Kitchin RM, Gadi IK, Smith BL, Sager R (1982): Genetic analysis of tumorigenesis: X. Chromosome studies of transformed mutants and tumor - derived CHEF18 cells. Somat Cell Genet 8:677-689. Simi S, Vatteroni L, Piras A, Mariani T, Rainaldi G (1990): Folate-sensitive fragile sites in Chinese hamster cell lines. Cancer Genet Cytogenet 46:209-216. Seabright MA (1971): A rapid banding technique for h u m a n chromosomes. Lancet ii:971-972. Ray M, Mohandas T (1976): Proposed banding nomenclature for the Chinese hamster chromosomes (Cricetulus griseus). Cytogenet Cell Genet 16:83-91. Mariani T (1989): Fragile sites and statistics. Hum Geuet 81:319-322. Gadi IK, Harrison JJ, Sager R (1984): Genetic analysis of tumorigenesis: XVI. Chromosome changes in azacytidine and insulin induced tumorigenesis. Somat Cell Mol Genet 10:521-529.
87
23. Kraemer PM, Ray FA, Bartholdi MF, Cram LS (1987): Spontaneous in vitro neoplastic evolution: selection of specific karyotypes in Chinese hamster cells. Cancer Genet Cytogenet 27:273-287. 24. Stalling RL, Crawford BD, Black RJ, Chang EH (1986): Assignment of RAS protooncogenes in Chinese hamster: implications for mammalian gene linkage conservation and neoplasia. Cytogenet Cell Genet 43:2-5. 25. Craig RW, Gadi IK, Sager R (1988): Genetic analysis of tumorigenesis: XXXI. Retention of short arm of chromosome 3 in suppressed CHEF hybrids containing c-Ha-ras (EJ) gene. Somat Cell Mol Genet 14:41-53. 26. Swindell JA, Ockey CH (1983): Cytogenetic changes during the early stages of liver carcinogenesis in Chinese hamster: an in vivo-in vitro comparison. Cancer Genet Cytogenet 8:23-36. 27. Cram LS, Bartholdi MF, Ray FA, Travis GI, Kraemer PM (1983): Spontaneous neoplastic evolution of Chinese hamster cells in culture: multistep progression of karyotype. Cancer Res 43:4828-4837. 28. Stenman G, Delorme EO, Lan CC, Sager R (1987): Transfection with plasmid pSV2gptEJ induces chromosome rearrangements in CHEF cells. Proc Natl Acad Sci USA 84:184-188. 29. Heim S, Mandahal N, Mitelman F (1988): Genetic convergence and divergence in tumor progression. Cancer Res 48:5911-5916. 30. Shin S, Freedman UH, Risser R, Pollack R (1975): Tumorigenicity of virus transformed cells in nude mice is correlated specifically with anchorage i n d e p e n d e n t growth in vitro. Proc Natl Acad Sci USA 11:4435-4439. 31. Suzuki K, Suzuki F, Watanabe M, Mikaido O (1989): Multistep nature of X-ray-induced neoplastic transformation in Golden hamster embryo cells: expression of transformed phenotypes and stepwise changes in karyotypes. Cancer Res 49:2134-2140. 32. Marshall CJ, Kitchiu RM, Sager R (19821: Genetic analysis ot tumorigenesis: XII. Genetic control of the anchorage requirement in CHEF cells. Somat Cell Genet 8:709-721. 33. Human Gene Mapping 10.5 (1990): Cytogenet Cell Genet: 55;358-386.
Cytogenetic changes were investigated during the spontaneous progression of CHEF18 Chinese hamster cells towards tumorigenicity. We farther report the chromosomal characterization of a series of spontaneous anchorage-independent clones, as well as of a series of tumor-derived cell lines resulting from injection of late passage cells in nude mice. The high karyotypic homogeneity (presence of four marker chromosomes strictly associated in all the metaphases analyzed) in all clones and tumorderived cell lines prompted us to alter this specific pattern of chromosomal aberrations in order to identify which if any of the aberrations were more strictly related to transformation. For this purpose we treated a tumor-derived cell line with Colcemid and analyzed the reversion of anchorage-independent phenotype in the subclones showing an altered association of the four m a r k e r chromosomes. We conclude that two of four marker chromosomes contribute to anchorage independence. ABSTRACT:
INTRODUCTION Most h u m a n [1, 2] and animal tumors [3, 4] are characterized by c h r o m o s m n a l aberrations. The findings of consistent c h r o m o s o m a l changes in m a n y hematopoietic malignancies [5] indicate that specific aberrations are involved in neoplastic transformation [6, 7]. However, the relationship between most of these aberrations and specific p h e n o t y p i c changes is still unclear, making it difficult to u n d e r s t a n d if they are causative or secondary events occurring during the neoplastic process. A n a p p r o a c h to investigate this problem is to examine cells at different steps of in vitro propagation, to determine if specific patterns of c h r o m o s o m a l changes can be observed and correlated to the expression of the transformed phenotype. CHEF 18, an immortalized, non-tumorigenic, fibroblastic Chinese hamster cell line [8], has been extensively used for genetic analysis of tumorigenesis [9, 10]. We have recently found that after prolonged serial passage this cell line undergoes s p o n t a n e o u s progression to tumorigenicity. This process is a c c o m p a n i e d by a r e d u c t i o n of proliferative heterogeneity [11] and increased ability both to grout in soft agar and to undergo gene amplification [12]. In this paper we report the karyotypic changes of CHEF18 cells during this progression and the chromosomal
From the Genetica e Biochimica Tossicologica, Istituto di Mutagenesi e Differenziamento CNR, Pisa, Italy. Address reprint requests to: Dr. Silvana Simi, IMD, Via Svezia 10, 56124, Pisa, Italy. Received February 24, 1992; accepted May 8, 1992. © 1992 Elsevier Science Publishing Co., Inc.
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characterization of a series of clones specifically selected at different stages of progression. All the selected clones showed only one specific and recurrent pattern of chromosomal changes c o m p r i s e d of four marker chromosomes. To determine the stringency of the four markers to the transformed p h e n o t y p e , we tried to obtain clones with different dosages of the four marker chromosomes. We have p r e v i o u s l y s h o w n that Colcemid, a microtubule disrupting agent k n o w n to i n d u c e p o l i p l o i d y [13] and chromosomal non disjunction [14], was also able to induce altered segregations [15] leading to n u m e r i c a l l y d i p l o i d cells, whose karyotypes presented a loss-gain of one chromosome and gain-loss of another [161. Thus we treated a t u m o r - d e r i v e d cell line with Colcemid and here report the c h r o m o s o m a l analysis of i n d u c e d subclones. Subclones were selected on the basis of altered segregations of the four marker c h r o m o s o m e s and challenged in soft agar. The results show that two markers cosegregated with the loss of anchorage i n d e p e n d e n c e . MATERIALS AND METHODS Cell Line
CHEF18 is an essentially d i p l o i d , untransformed, and nontumorigenic Chinese hamster cell line [8, 17]. This line was obtained by Prof. R. Sager (Dana-Farber Cancer Institute, Boston, MA) at passage 14. A standard protocol for propagation was used [11]. Briefly, the cells were c u l t u r e d in Dulbecco's Modified Eagle's m e d i u m s u p p l e m e n t e d with 10% fetal calf serum and in an a t m o s p h e r e of 6% CO2. Every 3 days the cells were t r y p s i n i z e d and a s a m p l e of 5 x 105 cells was propagated. With this schedule, an average ot four cell doublings occurred between two trypsinizations. 81 Cancer Genet Cytogenet 62:81 87 (1992) 0165-4608/92/$05.00
82
Cytogenetic Analysis E x p o n e n t i a l l y growing cells were treated with Colcemid, harvested, incubated with KC1 0.075 M, and fixed in methanol: acetic acid 3:1, as previonsly described [18]. Chromosome preparations were G-banded according to a trypsin digestion procedure [191. The proposed nomenclature of Ray and M o h a n d a s for Chinese hamster chromosomes was used [20].
Statistics A reliable statistical analysis using a basic program developed in our laboratory [211 was used to distinguish nonr a n d o m events from the casual a c c u m u l a t i o n of aberrations that can occur as a consequence of random distribution.
Soft-Agar Assay A n c h o r a g e - i n d e p e n d e n c e test was carried out as described elsewhere [12]. A p p r o p r i a t e concentrations of cells were cultured in semisolid agar m e d i u m and colonies were stained with cristal violet 3 weeks later. Only colonies equal or greater than 250 /am in diameter were counted. I n d i v i d u a l clones were isolated from soft-agar and chromosomal analysis was performed 6 - 1 0 days after isolation.
Tumorigenicity Assay Cells (8 × 106) were injected s u b c u t a n e o u s l y into 17 athymic Balb/C male and female mice. Tumors bigger than 5 m m in d i a m e t e r were scored as positive. Cell lines were established from eight i n d e p e n d e n t tumors and k a r y o t y p e d 6 - 1 0 days after isolation.
Colcemid Treatment Cultures were set up the day before the treatment at a density of 8 x 105 cells per 60-ram Petri dish. Colcemid (Boehringer, 0.025/xg/ml) was a d d e d for 16 hours. Following treatment, cells were w a s h e d and reincubated in fresh m e d i u m for a further 24 hours. Cells were then t r y p s i n i z e d and plated into 100-ram Petri dishes at 50,100, and 200 cells per dish. At 2 weeks, i n d i v i d u a l colonies were isolated using glass c y l i n d e r s coated with silicone lubricant and e x p a n d e d for c h r o m o s o m a l analysis.
RESULTS Karyotype Evolution We have a n a l y z e d the karyotypic evolution of CHEF18 cell line as it s p o n t a n e o u s l y became tumorigenic. The analysis of cells at different passages (Table 1) showed that in the early phases of in vitro propagation different karyotypes were generated and cells with an extra copy of the long arm of c h r o m o s o m e 3 (3q) and with a marker c h r o m o s o m e originating from c h r o m o s o m e 4 were significantly in excess. The structural alteration involving c h r o m o s o m e 4 gave rise to a rearranged c h r o m o s o m e 4 q + with a larger than normal long arm owing to insertion of an u n k n o w n segment distal to band 4q25. From the m i d d l e phase of propagation (after 60 days in culture), while the extra 3q is lost and 4 q + retained, a steady increase of karyotype instability occurred, which
S. Simi et al. finally resulted in the significant presence of other altered chromosomes originating essentially from c h r o m o s o m e s 7, 8, and 9. The structural alterations involving c h r o m o s o m e 7 consisted both of a c o m p l e t e deletion of the short arm generating a c h r o m o s o m e formed by the long arm of chromosome 7 (7q) and of the formation of a c h r o m o s o m e 7 p + : this may have occurred by centromeric fusion between 7q and another c h r o m o s o m a l element or by juxtaposition of a c h r o m o s o m a l segment (possibly from 8p, which is contemp o r a n e o u s l y deleted) on the short arm of 7. Because of the observation that when a 7q was present the 7 p + was not, and vice versa, and of the presence of a fragile site (18) at the centromere of c h r o m o s o m e 7, it seems likely that the marker 7p + represents a translocation of the whole short arm rather than a juxtaposition. However, the origin of chromosmnal material in 7p ÷ could not be determined, as the inserted material consisted of a single band. making identification impossible. Alterations involving c h r o m o s o m e 8 consisted of both c h r o m o s o m e 8q, with a c o m p l e t e l y deleted short arm, and, much more frequently, marker 8 p - , having a small deletion of the distal part of short arm at band 8p16. Finally, the structural alteration of c h r o m o s o m e 9 consisted of a marker 9q + , w h i c h seems to have a differentially staining amplified region, often of variable length, in the long arm. A metaphase presenting 4 q + , 7 p + , 8 p - . and 9q + marker c h r o m o s o m e s is s h o w n in Figure 1. The p o p u l a t i o n tended to r e m a i n d i p l o i d in all stages of progression, although a small fraction of s p o r a d i c a l l y occurring a n e u p l o i d cells was also present (data not shown).
Analysis of Anchorage-Independent Clones and Tumor.Derived Cell Lines A n c h o r a g e - i n d e p e n d e n t (AI) clones derived from m i d d l e passage cells seeded in soft agar and t u m o r - d e r i v e d cell lines resulting from injection of late passage cells in n u d e mice were cytogenetically a n a l y z e d to d e t e r m i n e if specific chromosomal changes correlated to both in vitro and in vivo transformation parameters. Tables 2 and 3, respectively, show the c h r o m o s o m a l changes found in five AI clones and in eight t u m o r - d e r i v e d cell lines. All clones and cell lines d i s p l a y e d a unique karyotypic pattern characterized by the simultaneous presence of four marker chromosomes, 4q + , 7p + , 8 p - , and 9q + , w h i c h were also observed, at various frequencies, during in vitro propagation.
Analysis of Clones and Suhclones Induced by Colcemid in 835T2 Tumor-Derived Cell Line To obtain clones with different dosage of marker chromosomes, 835T2 t u m o r - d e r i v e d cell line was treated with Colcemid. The G-banding cytogenetic analysis of 65 Colcemidi n d u c e d clones shows that eight clones d i s p l a y e d altered c h r o m o s o m e segregations, with different c h r o m o s o m e s r a n d o m l y involved in a n o m a l o u s segregations {Table 4). Of particular interest were clones colc 3/8 and colc 3/ 15, both presenting a deleted c h r o m o s o m e 7q originating by a centromeric break, one from marker c h r o m o s o m e 7p + {clone colc 3/8) and one from normal c h r o m o s o m e 7 (clone
Specific Chromosomal Aberrations and Transformation Table 1
83
Chromosome markers found at different intervals of in vitro propagation I n t e r v a l (in days)
Chromosome marker lp + lq lq 2p2q3p 3q 4q 4q+ 5q 5q÷ 6q 6q6q-
Band involved lp22 1ten lq22 2p28 2q31 3p16 3ten 4ten 4q25 5cen 5q23 6cen 6q16 6q24
7q } 7ten 7p+ 7q7q23 8p 8p16 8q 8cen 9q+ 9q22 Xq Xcen XqXq18 Yq Ycen Total m e t a p h a s e s analyzed
3
6
15
27
42
54
60
78
93
2
99
1
2
126
180
3
2
3
1 2
1 1
1 2
1 5 **~' 1 1
4** 10"**
1 4**
9**
6**
2
6***
8*** 1
2 1
2
1 4** 7***
1 1 2 14"**
1 1
36***
30***
1 2
2
3
1
1
1 2
1
1
4**
1
5***
1
2
1
5
1
1
2
45
40
40
40
1
2
36*** 1 2
4 15"**
4 24***
10"** 24***
7*** 3 5*
24*** 4 15"**
19"** 3 14"**
10"** 2 1 1
1 50
2 1
1
1
32***
40
6** 1 40
2
40
40
40
Cell s h o w i n g the p r e s e n c e of the four m a r k e r c h r o m o s o m e s .
~ ~i i¸¸~¸~¸
31"**
4
16"** 4 4 6*** 4 1 1
4 33***
2
21"** 13"** 12"** 2 14"**
2
" Asterisks indicate bands with a significant excess of lesions (*:p < 0.05: ** :P < 0,01; *** :p < 0.001).
Figure 1
105
q÷
7
t~
P÷
dsp_
40
40
40
84
Table 2
S. Simi et al.
Marker chromosomes found in five spontaneous a n c h o r a g e - i n d e p e n d e n t clones Marker t:hrolIlosome
Clone ~ 677/1 677/2 677:3 677:4 677/5
Metaphases analyzed
4q +
7p +
15 10 15 10 10
12 10 11 9 9
15 8 13 10 10
8p
9q
15 10 15 9 10
14 10 15 10 10
DISCUSSION
" All c l o n e s s h o w e d a d i p l o i d m o d a l c h r o m o s o l n e n u m b e r .
colc 3/15). Clone cole 1/6 is also interesting; it is aneuploid, having lost the normal copy of chromosome 7. To further disrupt the association of the four markers and to collect other clones with altered segregations, clone colc 3/8 was treated with Colcemid. The analysis of 28 Colcemid-induced subclones {see Table 5) identified four subclones displaying altered segregations. It is worth noting clone colc 4/23 lacking marker chromosome 8 p - and clone colc 4/69 lacking marker chromosome 9q +.
Anchorage-Independence Analysis in Clones and Subclones with Altered Segregations in Marker Chromosomes Table 6 shows the soft agar (SA) colony-forming ability of parental 835T2 tumor-derived cell line and of clones and subclones with altered segregation in the marker chromosonles.
While two of five clones were similar to the tumorderived cell line, three clones grew in soft agar with low efficiency. Table 7 shows the relative plating efficiency (RPE, the ratio of plating efficiency in soft agar to plating efficiency in liquid medium) correlated with the presence or absence of marker chromosomes. All chines and subclones retained the normal copy of chromosome 4 and the marker 4q +. When normal chromosome 7 is partially (clone colc 3/15) or completely lost (clone cole 1/16) growth in soft agar is not affected, whereas when the anomalous short arm of marker 7 p + is lost (clones colc 3/8 and 4/69) the ability to grow in soft agar is
Table 3
reduced. When, concurrently, marker 8 p - is also losl (clone cole 4/23) soft agar growth is greatly reduced. The presence or absence of marker 9 q + seemed u n i m p o r t a n t because two clones {cole 3/8 and 4/69), one with and the other lacking 9 q + , showed the same RPE. This analysis clearly shows that reversion from the anchorage-independent phenotype co-segregates with the loss of chromosomes 7p + and 8p - , whereas the presence or absence of chromosomes 4q + and 9q ~- did not affect soft agar growth.
In this stud), a karyotypic analysis of CHEF18 Chinese hamster cells during the spontaneous progression to tumorigenicity was carried out. CHEF18 Chinese hamster cells presented genetic instability during serial propagation; in addition to a broad spectrum of random chrmnosomal changes, there are n o n - r a n d o m alterations which are found to precede and then accompany the acquisition of tumorigenicity. In c o m m o n with other works [22, 23], an extra copy of the long arm of chromosome 3 {3q) (where two oncogenes have been mapped [24]), was the most frequent abnormality during the early steps of progression. Trisomy 3q, together with contemporary loss of the associated short arm (3p) (where the presence of a tumor suppressor gene has been suggested [25]), is supposed to be sufficient to induce tumorigenicity in Chinese hamster cells [22]. Our data suggest that cells with trisomy 3q had a selective advantage during the early phase of in vitro progression. Successively, n u m e r o u s other heteroploid karyotypes were generated, not necessarily all related to transformation, probably reflecting somatic events of the ongoing tumorigenic progression. Other chromosomes involved in the formation of markers were 4, 7, 8, and 9. One of the markers, 4q +, occurred very early during progression: the other three appear to occur later. Changes involving these chromosomes have been observed during transformation in Chinese hamster system; an extra copy of chromosome 4q was found during the early stages of liver carcinogenesis (26), whereas trisomy of chromosome 8, as well as the presence of 8q marker, were found during s p o n t a n e o u s neoplastic evolution of Chinese hamster cells 127].
Marker chromosomes found in eight tumor-derived cell lines Marker chromosome
(;ell line" ES1 ES2 835T1 835T2 835T3 835L4 834L5 834L6
Metaphases analyzed 10 10 6 11 10 9 11 13
2q -
3p -
3q
4q
2
1
3
1
" All cell lines showed a diploid modal chromosome number.
4q + 9 9 5 10 7 9 9 9
5q
6q --
1
1
7p t
8p -
9q +
9 10 6 11 10 9 11 13
9 10 6 11 10 9 12 1'3
1(1 10 5 11 9 9 i1 1'3
Xq
Yq
2 4 1 1 1
Specific C h r o m o s o m a l Aberrations and Transformation
Table 4
85
G-banding c h r o m o s o m a l analysis of clones i n d u c e d by Colcemid in 835T2 t u m o r d er i v ed cell line
A)" Clones with parental karyotype 51
Clones displaying altered segregations
Clones with hyper diploid or tetra ploid karyotype
Total clones analyzed
6
65
8
B)b Clone 835T2 (parental one) Colcl/5 Colc 1/6 Colcl/12 Colc 1/15 Colc 2/6 Colc 3/7 Colc 3/8 Colc 3/15
Modal chromosome number 22 25 21 22 (18-23) 24 22 22 22
Chromosomal constitution C -4+4q+,
-7+7p+,
-8+8p-,
-9+9q+,
-4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+,
-7+7p+, -7+7p+, -7+7p+, -7+7p+, -7+7p+, -7+7p+, -7p++7q, -7+7p+,
-8+8p-, -8+8p-, -8+8p , -8+8p-, -8+8p-, -8+8p-, -8+8p-, -8+8p-,
-9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+, -9+9q+,
+Y
+5,+9q, -7 -6+6q -4+4q +5,+Y -9+9q -7+7q
° A) Chromosomal constitution of all clones. ~'B] Detailed chromosomal constitution of the eight clones displaying altered segregations. ': Chromosome alterations were recorded when more than two thirds of the metaphases examined presented the alteration. Never less than 15 metaphases were analyzed per each clone.
In the CHEF system, the long arm of c h r o m o s o m e 4 was found differently rearranged in cells transfected with a plasmid [28], whereas m o n o s o m y of c h r o m oso m es 7 and/or 8 is one of the most consistent aberrations in transformed cells [25]. C h r o m o s o m e 9 with amplified regions was also sporadically found during azacytidine-induced tumorigenesis [22]. The breakpoints that form two of the observed rearranged c h r o m o s o m e s (3q and 7p + ) were in regions w h er e fragile sites are expressed; 3 cen and 7 cen have been reported to be folate-sensitive fragile sites in Chinese hamster
Table 5
c h r o m o s o m e s [18]. These data further em p h asi ze that fragile sites are predisposing factors for c h r o m o s o m a l changes and can contribute to the creation of stable c h r o m o s o m e rearrangements. In order to study the p h e n o t y p i c c o n s e q u e n c e s of these ch r o m o so m al changes, AI clones and t u m o r - d e r i v e d cell lines were analyzed. The data s h o w e d a constant presence of four marker chromosomes, w h i c h are u n i f o r m l y retained in all selected clones and t u m o r - d e r i v e d cell lines, indicating that transformation can occur w i t h o u t extensive genetic instability. Other karyotypes may have been generated, but,
G-banding c h r o m o s o m a l analysis of subclones i n d u ced by Colcemid in clone colc 3/8
A)~ Subclones with parental karyotype
Subclones displaying altered segregations
Subclones with hyper diploid or tetraploid karyotype
Total subclones analyzed
23
4
1
28
B) ~
Clone Colc 3/8 (parental one/ Colc 4/13 Colc 4/23 Colc 4/69 Colc 4/88
Modal chromosome number 22 21 Ca)21 b)22 a)(20-23) b)(21-23) 22
Chromosomal constitution ° -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q+, -4+4q,
°'~'see Table 4. ': Subpopulation a and b were present in approximately equal proportions.
7p+ +7q, -7p+ -7p+ -7p+ -7p+ -7p+ -7p+
+7q, +7q, +7q, +7q, +7q, +7q,
-8+8p-,
9+9q+
-8+8p-, -8p-, 8p , -8+8p-, -8+8p-, -8+8p-,
-9+9q+, -9+9q+ 9+9q+, -9+9q+ -9q+ -9+9q+,
-y +7
-X+Yq
86
Table 6
S. Simi et al.
Anchorage i n d e p e n d e m : e in 835T2 tumorderived cell line and in clones and subchmes displaying altered segregations of marker chromosomes
{2lone
P.E. in soft agar {%}
P.E. in DMEM (%}'
835T2 cole 3/15 col{: 1/6 col{: 3/8 cole 4/69 colc 4/23
51.2 41.9 37.5 7,4 7.3 1.1
100 100 100 61 60 49
" These results represent the average of two experimenls.
if so, they were lost, and one cannot e x c lu d e that further extensions of these studies could reveal other c h r o m o s o m e combinations that play a role in transformation and tumorigenicity. Different "scenarios" have been proposed for clonal evolution of neoplasms that can either evolve toward greater diversity (genetic divergence) or toward h o m o g e n ei t y (genetic convergence) [29]. U n d e r our standard culture conditions, the balance b e t w e e n genetic instability and selective pressure favored the emergence of a p r e d o m i n an t karyotype. To assess the functional role of the various c h r o mo so m al abnormalities found, C o l c e m i d - i n d u c e d clones with altered marker c h r o m o s o m e distribution were tested for their ability to grow in soft agar, because anchorage independence is generally regarded as the best tissue-culture correlate of tumorigenicity [30, 31]. The pattern of c h r o m o s o m e loss suggested that marker c h r o m o s o m e s 7p + and 8p - may be i n v o l v e d in transformation; clones w h i c h have lost either of these chromosomes grow poorly in soft agar, whereas the presence or the absence of 4q + and 9q + were without effect. It has been s h o w n [17, 32] that segregation of anchoragei n d e p e n d e n t subclones from somatic hybrids between anc h o r a g e - i n d e p e n d e n t and a n c h o r a g e - d e p e n d e n t CHEF cells was associated with the loss of all or part of one copy
Table 7
Relative plating efficiency (RPE] versus presence/ absence of marker c h r o m o s o m e s and their normal homologous Chromosome
Clone
RPE~'
835T2 Colc 3/15 Colc 1/6 Colc 3/8 Colc 4/69
51.2 41.9 37.5 12.1 12.1
Colc 4/23
2.2
~'see Results.
4 4, 4, 4, 4, 4, 4, 4, 4,
4q+ 4q+ 4q+ 4q+ 4q+ 4q+ 4q+ 4q+
7 7, 7p+ 7q, 7p+ 0, 7p+ 7, 7q 7, 7q 7, 7q 7, 7q 7, 7, 7q
8 8, 8, 8, 8, 8, 8, 8, 8,
8p8p8p8p8p8p0 O
9 9, 9, 9, 9, 9, 9, 9, 9,
9q+ 9q+ 9q+ 9q+ 0 9q+ 9q+ 9q+
of c h r o m o s o m e 1. In our work, however, we have found no significant alterations in c h r o m o s o m e 1. It appears that loss of genetic material from the short arm of c h r o m o s o m e 8 and loss or relocation of genetic material from the short arm of c h r o m o s o m e 7 may represent fundamental events in the spontaneous transformation of Chinese hamster cells, presumably conferring an altered growth requirement revealed as a n c h o r a g e - i n d e p e n d e n c e growth. The presence of specific c h r o m o s o m e aberrations is a signal that unidentified changes in the genome may have occurred. The observation that one alteration is a deletion and the other a translocation suggests that possible genetic changes could be specific allelic loss of suppressor genes and activation of protooncogenes. The fact that suppressor genes are supposed to be located on c h r o m o s o m e 7 and 8 [25] and that an oncogene, K-ras, has been m a p p e d on c h r o m o s o m e 8 [241, further supports the hypothesis. Tire other two genes m a p p e d on c h r o m o s o m e 8 are GAPD arrd TPY1, w h i c h are linked with K-ras. It is perhaps noteworthy that distal and terminal deletions of h o m o l o g o u s human 12p containing the same three genes are reported from various h u m a n n eo p l asm s [33]. The identification of other genes on c h r o m o s o m e 7 and 8 might help in understanding the relationship between c h r o m o s o m e aberrations, gene expression, and tumorigenic process in Chinese hamster sy'stem.
This work was partially supported by AIRC (Italian Association for Cancer Research) and CNR (National Research Council) Target Project "Applicazioni Cliniche della Ricerca Oncologica". Lucia Vatteroni is the recipient of a fellowship from AIRC. The authors thank Dr. E. Giulotto and Dr. M. Minks for critical review of the manuscript, and Mrs. G. Cecchi for typing.
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