Trans.Acfing Factors in Chromosomal Instability E. W. Campbell, D. Chen, J. Tesmer, R. Stallings, J. Longmire, and P. M. Kraemer
ABSTRACT: The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids, Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of humun-hamster-translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the C/-/X line that involve ongoing production of a diffusible clastogenic factor.
INTRODUCTION Genetic and c h r o m o s o m a l instability p l a y an important and perhaps essential role in the neoplastic process [1-3]. Evidence supporting this role has been accumulating for at least 80 years [3], yet what the defect in neoplastic cells might be in terms of i n v o l v e m e n t of cellular structures or specific genes is still not clear [4, 5]. In the present investigation, we a p p l i e d the methodology of somatic cell hybridization to determine whether trans-acting factors are involved in the chromosomal instability of Chinese hamster cell lines~ Transformed or immortalized Chinese hamster cell lines have been studied by us and other investigators for m a n y years [6-9], with the general conclusion that such lines arose by spontaneous neoplastic events that a c c u m u l a t e d during passage of the cells in culture. In almost all cases, cultured normal Chinese hamster cells have become immortalized, transformed, and tumorigenic, and these neoplastic changes have been associated with chromosome changes [10]. However, the degree of persistence of chromosomal instability are quite variable from line to line. On the one hand, some Chinese hamster lines, like the DON line, have e v i d e n t l y never been very unstable because even after years of culture they show only m o d e s t departures from a d i p l o i d karyotype. Other lines, of w h i c h CHO is a prime From the Los Alamos National Laboratory,Los Alainos, New Mexico Address reprint requests to: P. M..Kraemer, Ph.D., Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545. Received August 7, 1988; accepted December 27, 1988. 89 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:89-100 {1990) 0165-4608/90/$03.50
90
E . w . Campbell et al. example, are highly aneuploid, indicating chromosomal instability at some time in the past. Yet the CHO has been extraordinarily stable for many years, and all known CHO sublines and mutants share a majority of the 13 distinctive marker chromosomes first defined in 1973 [11, 12]. Finally, a few Chinese hamster lines have been reported [13, 14] that exhibit ongoing karyotypic instability; i.e., an instability characterized by ongoing production of new aberrant rearrangements and evidence of chromosome breakage. We have called one of the latter types of lines the CHX line [15]. In the present work, we compared the cytogenetic c o n s e q u e n c e s of hybridizing CHX and CHO cells to normal diploid human fibroblasts. In both cases, we concentrated our observations on the question of what happened to the human chromosome component as a function of passage in culture of the hybrid clones.
MATERIALS AND METHODS Cell Lines and Culture Conditions The Chinese hamster line we have designated CHX [15] was obtained from Dr. Penny Jeggo, M.R.C., Mill Hill, London. It was originally considered a CHO-K1 subline [16] although, as explained below, such a designation appears unlikely. A CHO line was also used in this study; this line has been maintained continuously in our laboratory since it was obtained from Dr. Ted Puck in •962. A normal human skin fibroblastic cell strain, HSF 24, was established according to the method described by Noyes et al. [17]. All cells were cultured in a-minimum essential medium (a-MEM) supplemented with 100/,~g/ml streptomycin, 100 U/ml penicillin, and 10% fetal calf serum. The cultures were maintained at 37°C in a humidified incubator with 5% CO2 tension. Chinese hamster/human hybrid lines were prepared using spontaneous ouabain and 6-thioguanine double-resistant mutants derived from the two Chinese hamster lines (CHO and CHX). These were fused with the normal human fibroblasts, passage 7, with polyethylene glycol (PEG 1000, Baker Phillipsburg, NJ) according to the method of Davidson et al. [18], and hybrids were selected with medium containing ouabain (10 3M) and hypoxanthine, aminopterin, thymidine (HAT) [19]. Individual colonies were isolated and expanded, and their chromosome content was identified.
Chromosome Analyses Metaphase spreads of the hybrid cells were prepared from trypsinized monolayer cultures treated with Colcemid (0.1/~g/ml) for 1-2 hours. The cells were swollen in hypotonic KC1 solution, fixed in methanol:acetic acid (3:1 ml/ml), and dropped onto cold wet microscope slides. The slides were stained by a fluorescence in situ hybridization procedure [20] using biotin-labeled human genomic DNA as probe. Slides were counterstained with propidium iodide (1.0/~g/ml) and analyzed as wet mounts by fluorescence microscopy. Analyses of parental cells by G banding were described previously [15].
DNA Fingerprinting Tandem-repetitive DNA sequences can be used to establish the individual identity of somatic cells [21]. Our methods use probes [22] derived from sequences first identified in the M13 phage [23]. These methods have been described previously [241; genomic DNAs from cell cultures were isolated, restricted with Hae III or Hinf I, and electrophoresed in a 0.7% agarose gel at 3.5 V/cm for about 36 hours. The separated DNA
91
Trans-Acting Factors in Chromosomal Instability
CHO
D
4 1
2
X
57
88910
2
3
° 45
678
9 i0 12 13
"Z" C H R O M O S O M E S
CHX
I
1
2
2 3
8
A B
C
D
E F
G H
MARKER AND ABBERENT
I
u u
u u u u
CHROMOSOMES
Figure 1 G-Banded karyotypes of a C H O cell and a C H X cell, both with 21 chromosomes [from reference 15, republished with permmsion, Karger, Basel].
fragments were transferred to nylon filters (Zetabind, Cuno Meriden, Co) and hybridized with variable-number-tandem-repeat (VNTR) probes labeled by nick translation. Hybridization conditions were as described by Vassart et al. [23]. After washes at moderate stringency (I x SSC, 0.1% sodium dodecyl sulfate at 65°C), the filters were exposed to Kodak XAR-5 film at - 70°C with intensifying screens. RESULTS Characteristics of Parental Cells
The hybrid cell lines were derived by fusion of human diploid fibroblasts (HSF 24), to either CHO cells or CHX cells. The two Chinese hamster parental cell lines were selected because they were strikingly different in the stability of their karyotypes [15]. Although both lines are aneuploid and tumorigenic in nude mice, the CHO line has retained a fairly homogenous karyotype since it was first defined in 1973 [11]. In contrast, the CHX line consists of cells possessing one or more copies of four normalappearing chromosomes (# 1, 2, 3, and 8), nine marker chromosomes; in addition, each cell has between several and many uniquely rearranged chromosomes (Fig.
92
E.W. Campbell et al.
CHROMOSOME NUMBER DISTRIBUTIONS 42
I
I
!
36
I
I
C l i O / 1 2 5 : 4 9 CELLS
30 24 18 12 6 I
I
I
I
20
25
30
35
40
45
50
!
I
I
I
I
I
I
0
15 15
CHX:
==
I
1 12CELLS
lO
0 15
20
25
30
35
40
45
50 and
Figure 2 Chromosome number distributions of CHO and CHX parental cells used in this study.
1). The total number of chromosomes in CHX cells varies between subdiploid to hypertetraploid without any clear modal stem numbers (Fig. 2). Other evidence of an ongoing destabilizing process included a high incidence of mitotic spreads with acentric fragments and unstable dicentric chromosomes. However, we did not find a significantly elevated rate of sister chromatid exchanges in the CHX cells (Table 1). The provenance of the CHX cell line is not clear. At the time we obtained these cells, they were labeled CHO K1. However, as shown in Figure 1, although the CHX cells were clearly of Chinese hamster origin, they had none of the characteristic "Z" chromosomes of CHO cells [11]. They also had an apparently normal chromosome 3, which CHO cells lack [11]. We explored this discrepency using DNA sequence probes derived from VNTRs. Such probes have been used for individual identification of humans and other mammals as well as for establishing the provenance of cell lines [25-27]. Figure 3 shows such analyses using DNA from CHO and CHX cells as well as five subclones and three recultured tumors from CHX. The nine CHX sublines show a few band differences among them, reflecting changes consistent with the karyotypic instability and heterogeneity of neoplastic cells [24, 28, 29]. The CHO
93
T r a n s - A c t i n g Factors in C h r o m o s o m a l Instability
Table 1
Sister C h r o m a t i d E x c h a n g e s in CHO and CHX cell CHO cells
CHX cells
Cell no.
No. of CHR
No. of SCE
No. of CHR
No. of SCE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Totals SCE/CHR
21 20 21 21 21 21 20 20 20 20 18 20 21 21 19 20 21 22 21 22 20 20 19 21 19 509
4 10 5 6 5 1 4 8 9 5 9 4 7 5 6 6 9 7 6 9 4 4 4 2 7 146
36 21 19 15 21 17 24 13 20 23 22 15 39 24 20 26 21 15 18 19 19 18 19 15 19 518
10 6 1 4 4 4 12 6 5 6 4 7 12 9 4 4 11 4 5 4 10 6 6 5 13 162
0.29
0.31
Abbreviation: SCE, sister chromatid exchange.
b a n d pattern clearly h a d several differences from all of the CHX lineages but n e v e r t h e less h a d c o n s i d e r a b l e r e s e m b l a n c e to the latter, as p r e v i o u s l y r e p o r t e d by T h a c k e r et al. [27].
Chromosome Changes in Human/Hamster Clones After cell f u s i o n and selection, c o l o n i e s w e r e e x p a n d e d to about 20 d o u b l i n g s (106 cells) and assayed for the p r e s e n c e of b o t h h u m a n and C h i n e s e h a m s t e r c h r o m o s o m e s . T h r e e c l o n e s w e r e s e l e c t e d for d e t a i l e d analysis at that d o u b l i n g l e v e l and w e r e also carried in c u l t u r e for a d d i t i o n a l analyses after about 60 d o u b l i n g s and 100 doublings. T h e three lineages c o n s i s t e d of o n e typical CHO x H S F h y b r i d line d e s i g n a t e d HOi and t w o CHX × H S F lines d e s i g n a t e d HX5/3-7 and HX7/1. All three lines c o n t i n u e d to lose h u m a n c h r o m o s o m e s t h r o u g h o u t the e x p e r i m e n t , but all three r e t a i n e d at least a few h u m a n c h r o m o s o m e s w h e n e x a m i n e d after 100 d o u b l i n g s {Table 2). In a d d i t i o n , all three lines r e t a i n e d excess n u m b e r s of C h i n e s e h a m s t e r c h r o m o s o m e s and there was c o n s i d e r a b l e n u m e r i c a l h e t e r o g e n e i t y in e a c h case. H e n c e , n u m e r i c a l instability c o u l d n o t be s h o w n to be a f u n c t i o n of the particular C h i n e s e h a m s t e r b a c k g r o u n d in these experiments. Table 3 s h o w s the o c c u r r e n c e and stability of t r a n s l o c a t i o n s b e t w e e n h u m a n a n d C h i n e s e h a m s t e r c h r o m o s o m e s . S u c h t r a n s l o c a t i o n s w e r e i n f r e q u e n t in the C H O x
94
E . w . Campbell et el,
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3
4
5 T1 T 2 T 3
Kb 23.1
i
qgai~ " - - -
~
~
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9.4
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D
alma
i
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4.4
Figure 3 Restriction patterns of DNA from CHO and CHX cells, digested with HinfI, electrophoresed, transferred, and probed with p47.2 [22]. Parental cells were used for the first two lanes; lanes 1-4 represent CHX subclones and lanes T1-T3 represent tumors recultured from nude mice inoculated with 106 CHX cells. h u m a n d i p l o i d fibroblast line designated HO-1 as well as in other similar lines (data n o w shown). Only two h u m a n - h a m s t e r - t r a n s l o c a t e d marker chromosomes (Figs. 4 and 5) were noted during the observation period, and these two might be stable markers since at 100 doublings they were found in about one third of cells examined. In contrast, the two cell lines from the CHX parent had m a n y translocations involving h u m a n chromosomes (Fig. 5). These hybrid translocations occurred throughout the observation period, and m a n y of them appeared to be very unstable, appearing only in a single cell. Some of the latter were dicentric chromosomes, w h i c h perhaps w o u l d explain their instability. The HX5/3-7 line also showed three early marker chromosomes and these three persisted throughout the observation period. Direct evidence of the clastogenicity of the CHX background on h u m a n chromo-
95
Trans-Acting Factors in Chromosomal Instability
Table 2
Chromosome Numbers in hybrid clones ° as a function of passage in culture Doublings in culture
Cell line HO-1 Human Hamster Human-hamster HX5/3-7 Human Hamster Human-hamster HX7/1 Human Hamster Human-hamster
20
60
100
10 (6-15) 34 (20-40) 0 (0)
9 (6-12) 32 (23-39) 0 (0)
8 (2-11) 30 (20-38) 0.73 (0-2)
3 (0-6) 51 (19-75) 1.23 (0-3)
2 (0-3) 57 (18-86) 0.87 (0-3)
2 (1-4) 51 (40-76) 0,75 (0-2)
16 (9-36) 31 (21-60) 0.40 (0-2)
12 (5-24) 30 (23-45) 0.50 (0-2)
6 (3-17) 31 (21-51) 0.81 (0-3)
° Mean (range) per cell; 15-31 cells were scored for each cell population. somes was p r o v i d e d by the presence of h u m a n acentric fragments and/or minutes. Many of the CHX-human hybrid cells had one or two and occasionally up to 10 h u m a n acentric fragments and often hamster acentric fragments as well. Table 4 shows the data as a percentage of cells with one or more h u m a n acentric fragments. Their occurrence is clearly a function of the CHX background and appears to be progressive with time. In addition, about 10% of the CHX-human hybrid cells had one or two fragmented or severely distorted chromosomes, either hamster or h u m a n or both.
DISCUSSION In the present study, we tried to exploit differences in the chromosomal stability of two Chinese hamster cell lines to determine whether trans-acting elements might play a role in generation of chromosome instability. Early in its culture history, the CHO line was very unstable and u n d e r w e n t many chromosomal rearrangements as part of the spontaneous neoplastic process that occurs w h e n Chinese hamster cells are grown in culture [30]. That process reached some sort of stable state m a n y years ago, and the line became almost as stable as nonneoplastic cell strains. In contrast, an active clastogenic and numerically destabilizing process is still occurring in the CHX line. The active nature of the process is indicated by the extremely variable c h r o m o s o m e number in freshly cloned ceils and in the occurrence of aberrations such as acentric fragments and unstable dicentric and other rearrangements. The question posed was w h e t h e r chromosomes from normal h u m a n d i p l o i d fibroblasts w o u l d be differentially affected in the two kinds of Chinese hamster backgrounds. In these different types of hybrids, the h u m a n chromosome component, including microscopically visible blocks of translocated h u m a n DNA, were followed for the first 100 doublings after cell fusion. The occurrence of chromosomes with hamster and h u m a n material translocated into a single chromosome was used as a measure of the destabilizing effect of the hamster background. Although the two hamster lines differ in both structure and numerical chromosomal stability, numerical instability is also i n t r o d u c e d by the cell hybridization procedure. Hence, in the present study, only structural instability was measurable. Several aspects of the CHX instability involved
96
E . W . Campbell et al.
Table 3
Occurrence of h u m a n - h a m s t e r - t r a n s l o c a t e d c h r o m o s o m e s as a function of passage in culture Doublings in culture
Human-hamstertranslocated chromosome a HO-1 A B HX5/3-7 A B C D E F G H HX7/1 A B C D E F G H I J K L M N O P Q
20
60
100
0/15 0/15
5/15 6/15
11/31 8/31 13/31 3/31 3/31 0/31 0/31 0/31
2/16 2/16 6/16 0/16 0/16 2/16 1/16 1/16
2/16 5/16 2/16 0/16 0/16 1/16 0/16 2/16
1/30 11/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30
0/16 0/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 0/16 0/16 0/16 0/16 0/16 0/16
0/16 0/16 1/16 0/16 2/16 0/16 0/16 2/16 0/16 0/16 0/16 1/16 2/16 2/16 1/16 1/16 1/16
0/15 b 0/15
° Human-hamster-translocated chromosomes are diagramed in Figure 5. bNumber observed over number of metaphases examined.
the h u m a n c h r o m o s o m e c o m p o n e n t in the CHX-human hybrids. However, the results s h o wn in Table 2, as well as other data not shown, indicate that the general patterns of h u m a n c h r o m o s o m e retention and loss were highly variable from line to line wi t h o u t any large d e p e n d e n c y on the particular Chinese hamster background. Humanh a m s t e r - t r a n s l o c a t e d chromosomes occurred more frequently in the CHX-human hybrids than in those with CHO background, and they tended to be transient. In addition, the frequent occurrence of h u m a n acentric fragments and the occasional observation of mixtures of broken h u m a n and Chinese hamster fragments in the CHX hybrid strongly support a dominant heritable role for some aspect of the CHX background. The results are consistent with the ongoing production in the CHX line of a trans-acting clastogenic factor. Such a factor has been reported as being i n d u c e d by ultraviolet (UV) irradiation and contributing to chromosome aberrations at the first mitosis [31]. Unlike the present study, however, the indirect UV irradiation effect
Trans-Acting Factors in Chromosomal Instability
97
Figure 4 Metaphase spread from the HO-1 hybrid clone after 100 doublings in culture. The two human hamster translocated chromosomes were present in about one third of the cells.
included a modest increase in sister chromatid exchanges [32]. A diffusible factor has also been reported after a-irradiation of Chinese hamster embryo cells transformed with SV40 [33]. In this case, the factor induced amplification of integrated SV40 sequences, a result that could involve production of acentric fragments [34]. T Antigen itself can also be considered a diffusible clastogenic factor; we recently showed that human diploid fibroblasts with an integrated SV40 A gene are mainly driven toward neoplastic transformation by ongoing clastogenicity and selection [35]. Although our data are consistent with a trans-acting factor, other possibilities have not been ruled ont. For instance, if the high cytogenetic instability of the CHX line is primarily a consequence of a heritably defective structure such as nuclear matrix or mitotic spindle [5], the CHX hybrids might have the same problem in maintaining stability of the chromosomes from either parent. The list of gene products that might have a role in chromosomal stability is very long indeed [4]; however, little direct evidence now exists to indicate which of them is(are) important in neoplasia. The consistent association of numerical and structural chromosomal changes in preneo-
98
E . w . Campbell et al.
DIAGRAMS HUMAN-HAMSTER
OF
HYBRID
CHROMOSOMES
Ho-1
Hx7/1 A
A
B
B C
Hx5/3-7
B
D E F
C
G
D E F
H I J
G 0
ABSTRACT: The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids, Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of humun-hamster-translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the C/-/X line that involve ongoing production of a diffusible clastogenic factor.
INTRODUCTION Genetic and c h r o m o s o m a l instability p l a y an important and perhaps essential role in the neoplastic process [1-3]. Evidence supporting this role has been accumulating for at least 80 years [3], yet what the defect in neoplastic cells might be in terms of i n v o l v e m e n t of cellular structures or specific genes is still not clear [4, 5]. In the present investigation, we a p p l i e d the methodology of somatic cell hybridization to determine whether trans-acting factors are involved in the chromosomal instability of Chinese hamster cell lines~ Transformed or immortalized Chinese hamster cell lines have been studied by us and other investigators for m a n y years [6-9], with the general conclusion that such lines arose by spontaneous neoplastic events that a c c u m u l a t e d during passage of the cells in culture. In almost all cases, cultured normal Chinese hamster cells have become immortalized, transformed, and tumorigenic, and these neoplastic changes have been associated with chromosome changes [10]. However, the degree of persistence of chromosomal instability are quite variable from line to line. On the one hand, some Chinese hamster lines, like the DON line, have e v i d e n t l y never been very unstable because even after years of culture they show only m o d e s t departures from a d i p l o i d karyotype. Other lines, of w h i c h CHO is a prime From the Los Alamos National Laboratory,Los Alainos, New Mexico Address reprint requests to: P. M..Kraemer, Ph.D., Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545. Received August 7, 1988; accepted December 27, 1988. 89 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:89-100 {1990) 0165-4608/90/$03.50
90
E . w . Campbell et al. example, are highly aneuploid, indicating chromosomal instability at some time in the past. Yet the CHO has been extraordinarily stable for many years, and all known CHO sublines and mutants share a majority of the 13 distinctive marker chromosomes first defined in 1973 [11, 12]. Finally, a few Chinese hamster lines have been reported [13, 14] that exhibit ongoing karyotypic instability; i.e., an instability characterized by ongoing production of new aberrant rearrangements and evidence of chromosome breakage. We have called one of the latter types of lines the CHX line [15]. In the present work, we compared the cytogenetic c o n s e q u e n c e s of hybridizing CHX and CHO cells to normal diploid human fibroblasts. In both cases, we concentrated our observations on the question of what happened to the human chromosome component as a function of passage in culture of the hybrid clones.
MATERIALS AND METHODS Cell Lines and Culture Conditions The Chinese hamster line we have designated CHX [15] was obtained from Dr. Penny Jeggo, M.R.C., Mill Hill, London. It was originally considered a CHO-K1 subline [16] although, as explained below, such a designation appears unlikely. A CHO line was also used in this study; this line has been maintained continuously in our laboratory since it was obtained from Dr. Ted Puck in •962. A normal human skin fibroblastic cell strain, HSF 24, was established according to the method described by Noyes et al. [17]. All cells were cultured in a-minimum essential medium (a-MEM) supplemented with 100/,~g/ml streptomycin, 100 U/ml penicillin, and 10% fetal calf serum. The cultures were maintained at 37°C in a humidified incubator with 5% CO2 tension. Chinese hamster/human hybrid lines were prepared using spontaneous ouabain and 6-thioguanine double-resistant mutants derived from the two Chinese hamster lines (CHO and CHX). These were fused with the normal human fibroblasts, passage 7, with polyethylene glycol (PEG 1000, Baker Phillipsburg, NJ) according to the method of Davidson et al. [18], and hybrids were selected with medium containing ouabain (10 3M) and hypoxanthine, aminopterin, thymidine (HAT) [19]. Individual colonies were isolated and expanded, and their chromosome content was identified.
Chromosome Analyses Metaphase spreads of the hybrid cells were prepared from trypsinized monolayer cultures treated with Colcemid (0.1/~g/ml) for 1-2 hours. The cells were swollen in hypotonic KC1 solution, fixed in methanol:acetic acid (3:1 ml/ml), and dropped onto cold wet microscope slides. The slides were stained by a fluorescence in situ hybridization procedure [20] using biotin-labeled human genomic DNA as probe. Slides were counterstained with propidium iodide (1.0/~g/ml) and analyzed as wet mounts by fluorescence microscopy. Analyses of parental cells by G banding were described previously [15].
DNA Fingerprinting Tandem-repetitive DNA sequences can be used to establish the individual identity of somatic cells [21]. Our methods use probes [22] derived from sequences first identified in the M13 phage [23]. These methods have been described previously [241; genomic DNAs from cell cultures were isolated, restricted with Hae III or Hinf I, and electrophoresed in a 0.7% agarose gel at 3.5 V/cm for about 36 hours. The separated DNA
91
Trans-Acting Factors in Chromosomal Instability
CHO
D
4 1
2
X
57
88910
2
3
° 45
678
9 i0 12 13
"Z" C H R O M O S O M E S
CHX
I
1
2
2 3
8
A B
C
D
E F
G H
MARKER AND ABBERENT
I
u u
u u u u
CHROMOSOMES
Figure 1 G-Banded karyotypes of a C H O cell and a C H X cell, both with 21 chromosomes [from reference 15, republished with permmsion, Karger, Basel].
fragments were transferred to nylon filters (Zetabind, Cuno Meriden, Co) and hybridized with variable-number-tandem-repeat (VNTR) probes labeled by nick translation. Hybridization conditions were as described by Vassart et al. [23]. After washes at moderate stringency (I x SSC, 0.1% sodium dodecyl sulfate at 65°C), the filters were exposed to Kodak XAR-5 film at - 70°C with intensifying screens. RESULTS Characteristics of Parental Cells
The hybrid cell lines were derived by fusion of human diploid fibroblasts (HSF 24), to either CHO cells or CHX cells. The two Chinese hamster parental cell lines were selected because they were strikingly different in the stability of their karyotypes [15]. Although both lines are aneuploid and tumorigenic in nude mice, the CHO line has retained a fairly homogenous karyotype since it was first defined in 1973 [11]. In contrast, the CHX line consists of cells possessing one or more copies of four normalappearing chromosomes (# 1, 2, 3, and 8), nine marker chromosomes; in addition, each cell has between several and many uniquely rearranged chromosomes (Fig.
92
E.W. Campbell et al.
CHROMOSOME NUMBER DISTRIBUTIONS 42
I
I
!
36
I
I
C l i O / 1 2 5 : 4 9 CELLS
30 24 18 12 6 I
I
I
I
20
25
30
35
40
45
50
!
I
I
I
I
I
I
0
15 15
CHX:
==
I
1 12CELLS
lO
0 15
20
25
30
35
40
45
50 and
Figure 2 Chromosome number distributions of CHO and CHX parental cells used in this study.
1). The total number of chromosomes in CHX cells varies between subdiploid to hypertetraploid without any clear modal stem numbers (Fig. 2). Other evidence of an ongoing destabilizing process included a high incidence of mitotic spreads with acentric fragments and unstable dicentric chromosomes. However, we did not find a significantly elevated rate of sister chromatid exchanges in the CHX cells (Table 1). The provenance of the CHX cell line is not clear. At the time we obtained these cells, they were labeled CHO K1. However, as shown in Figure 1, although the CHX cells were clearly of Chinese hamster origin, they had none of the characteristic "Z" chromosomes of CHO cells [11]. They also had an apparently normal chromosome 3, which CHO cells lack [11]. We explored this discrepency using DNA sequence probes derived from VNTRs. Such probes have been used for individual identification of humans and other mammals as well as for establishing the provenance of cell lines [25-27]. Figure 3 shows such analyses using DNA from CHO and CHX cells as well as five subclones and three recultured tumors from CHX. The nine CHX sublines show a few band differences among them, reflecting changes consistent with the karyotypic instability and heterogeneity of neoplastic cells [24, 28, 29]. The CHO
93
T r a n s - A c t i n g Factors in C h r o m o s o m a l Instability
Table 1
Sister C h r o m a t i d E x c h a n g e s in CHO and CHX cell CHO cells
CHX cells
Cell no.
No. of CHR
No. of SCE
No. of CHR
No. of SCE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Totals SCE/CHR
21 20 21 21 21 21 20 20 20 20 18 20 21 21 19 20 21 22 21 22 20 20 19 21 19 509
4 10 5 6 5 1 4 8 9 5 9 4 7 5 6 6 9 7 6 9 4 4 4 2 7 146
36 21 19 15 21 17 24 13 20 23 22 15 39 24 20 26 21 15 18 19 19 18 19 15 19 518
10 6 1 4 4 4 12 6 5 6 4 7 12 9 4 4 11 4 5 4 10 6 6 5 13 162
0.29
0.31
Abbreviation: SCE, sister chromatid exchange.
b a n d pattern clearly h a d several differences from all of the CHX lineages but n e v e r t h e less h a d c o n s i d e r a b l e r e s e m b l a n c e to the latter, as p r e v i o u s l y r e p o r t e d by T h a c k e r et al. [27].
Chromosome Changes in Human/Hamster Clones After cell f u s i o n and selection, c o l o n i e s w e r e e x p a n d e d to about 20 d o u b l i n g s (106 cells) and assayed for the p r e s e n c e of b o t h h u m a n and C h i n e s e h a m s t e r c h r o m o s o m e s . T h r e e c l o n e s w e r e s e l e c t e d for d e t a i l e d analysis at that d o u b l i n g l e v e l and w e r e also carried in c u l t u r e for a d d i t i o n a l analyses after about 60 d o u b l i n g s and 100 doublings. T h e three lineages c o n s i s t e d of o n e typical CHO x H S F h y b r i d line d e s i g n a t e d HOi and t w o CHX × H S F lines d e s i g n a t e d HX5/3-7 and HX7/1. All three lines c o n t i n u e d to lose h u m a n c h r o m o s o m e s t h r o u g h o u t the e x p e r i m e n t , but all three r e t a i n e d at least a few h u m a n c h r o m o s o m e s w h e n e x a m i n e d after 100 d o u b l i n g s {Table 2). In a d d i t i o n , all three lines r e t a i n e d excess n u m b e r s of C h i n e s e h a m s t e r c h r o m o s o m e s and there was c o n s i d e r a b l e n u m e r i c a l h e t e r o g e n e i t y in e a c h case. H e n c e , n u m e r i c a l instability c o u l d n o t be s h o w n to be a f u n c t i o n of the particular C h i n e s e h a m s t e r b a c k g r o u n d in these experiments. Table 3 s h o w s the o c c u r r e n c e and stability of t r a n s l o c a t i o n s b e t w e e n h u m a n a n d C h i n e s e h a m s t e r c h r o m o s o m e s . S u c h t r a n s l o c a t i o n s w e r e i n f r e q u e n t in the C H O x
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Figure 3 Restriction patterns of DNA from CHO and CHX cells, digested with HinfI, electrophoresed, transferred, and probed with p47.2 [22]. Parental cells were used for the first two lanes; lanes 1-4 represent CHX subclones and lanes T1-T3 represent tumors recultured from nude mice inoculated with 106 CHX cells. h u m a n d i p l o i d fibroblast line designated HO-1 as well as in other similar lines (data n o w shown). Only two h u m a n - h a m s t e r - t r a n s l o c a t e d marker chromosomes (Figs. 4 and 5) were noted during the observation period, and these two might be stable markers since at 100 doublings they were found in about one third of cells examined. In contrast, the two cell lines from the CHX parent had m a n y translocations involving h u m a n chromosomes (Fig. 5). These hybrid translocations occurred throughout the observation period, and m a n y of them appeared to be very unstable, appearing only in a single cell. Some of the latter were dicentric chromosomes, w h i c h perhaps w o u l d explain their instability. The HX5/3-7 line also showed three early marker chromosomes and these three persisted throughout the observation period. Direct evidence of the clastogenicity of the CHX background on h u m a n chromo-
95
Trans-Acting Factors in Chromosomal Instability
Table 2
Chromosome Numbers in hybrid clones ° as a function of passage in culture Doublings in culture
Cell line HO-1 Human Hamster Human-hamster HX5/3-7 Human Hamster Human-hamster HX7/1 Human Hamster Human-hamster
20
60
100
10 (6-15) 34 (20-40) 0 (0)
9 (6-12) 32 (23-39) 0 (0)
8 (2-11) 30 (20-38) 0.73 (0-2)
3 (0-6) 51 (19-75) 1.23 (0-3)
2 (0-3) 57 (18-86) 0.87 (0-3)
2 (1-4) 51 (40-76) 0,75 (0-2)
16 (9-36) 31 (21-60) 0.40 (0-2)
12 (5-24) 30 (23-45) 0.50 (0-2)
6 (3-17) 31 (21-51) 0.81 (0-3)
° Mean (range) per cell; 15-31 cells were scored for each cell population. somes was p r o v i d e d by the presence of h u m a n acentric fragments and/or minutes. Many of the CHX-human hybrid cells had one or two and occasionally up to 10 h u m a n acentric fragments and often hamster acentric fragments as well. Table 4 shows the data as a percentage of cells with one or more h u m a n acentric fragments. Their occurrence is clearly a function of the CHX background and appears to be progressive with time. In addition, about 10% of the CHX-human hybrid cells had one or two fragmented or severely distorted chromosomes, either hamster or h u m a n or both.
DISCUSSION In the present study, we tried to exploit differences in the chromosomal stability of two Chinese hamster cell lines to determine whether trans-acting elements might play a role in generation of chromosome instability. Early in its culture history, the CHO line was very unstable and u n d e r w e n t many chromosomal rearrangements as part of the spontaneous neoplastic process that occurs w h e n Chinese hamster cells are grown in culture [30]. That process reached some sort of stable state m a n y years ago, and the line became almost as stable as nonneoplastic cell strains. In contrast, an active clastogenic and numerically destabilizing process is still occurring in the CHX line. The active nature of the process is indicated by the extremely variable c h r o m o s o m e number in freshly cloned ceils and in the occurrence of aberrations such as acentric fragments and unstable dicentric and other rearrangements. The question posed was w h e t h e r chromosomes from normal h u m a n d i p l o i d fibroblasts w o u l d be differentially affected in the two kinds of Chinese hamster backgrounds. In these different types of hybrids, the h u m a n chromosome component, including microscopically visible blocks of translocated h u m a n DNA, were followed for the first 100 doublings after cell fusion. The occurrence of chromosomes with hamster and h u m a n material translocated into a single chromosome was used as a measure of the destabilizing effect of the hamster background. Although the two hamster lines differ in both structure and numerical chromosomal stability, numerical instability is also i n t r o d u c e d by the cell hybridization procedure. Hence, in the present study, only structural instability was measurable. Several aspects of the CHX instability involved
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Table 3
Occurrence of h u m a n - h a m s t e r - t r a n s l o c a t e d c h r o m o s o m e s as a function of passage in culture Doublings in culture
Human-hamstertranslocated chromosome a HO-1 A B HX5/3-7 A B C D E F G H HX7/1 A B C D E F G H I J K L M N O P Q
20
60
100
0/15 0/15
5/15 6/15
11/31 8/31 13/31 3/31 3/31 0/31 0/31 0/31
2/16 2/16 6/16 0/16 0/16 2/16 1/16 1/16
2/16 5/16 2/16 0/16 0/16 1/16 0/16 2/16
1/30 11/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30
0/16 0/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 1/16 0/16 0/16 0/16 0/16 0/16 0/16
0/16 0/16 1/16 0/16 2/16 0/16 0/16 2/16 0/16 0/16 0/16 1/16 2/16 2/16 1/16 1/16 1/16
0/15 b 0/15
° Human-hamster-translocated chromosomes are diagramed in Figure 5. bNumber observed over number of metaphases examined.
the h u m a n c h r o m o s o m e c o m p o n e n t in the CHX-human hybrids. However, the results s h o wn in Table 2, as well as other data not shown, indicate that the general patterns of h u m a n c h r o m o s o m e retention and loss were highly variable from line to line wi t h o u t any large d e p e n d e n c y on the particular Chinese hamster background. Humanh a m s t e r - t r a n s l o c a t e d chromosomes occurred more frequently in the CHX-human hybrids than in those with CHO background, and they tended to be transient. In addition, the frequent occurrence of h u m a n acentric fragments and the occasional observation of mixtures of broken h u m a n and Chinese hamster fragments in the CHX hybrid strongly support a dominant heritable role for some aspect of the CHX background. The results are consistent with the ongoing production in the CHX line of a trans-acting clastogenic factor. Such a factor has been reported as being i n d u c e d by ultraviolet (UV) irradiation and contributing to chromosome aberrations at the first mitosis [31]. Unlike the present study, however, the indirect UV irradiation effect
Trans-Acting Factors in Chromosomal Instability
97
Figure 4 Metaphase spread from the HO-1 hybrid clone after 100 doublings in culture. The two human hamster translocated chromosomes were present in about one third of the cells.
included a modest increase in sister chromatid exchanges [32]. A diffusible factor has also been reported after a-irradiation of Chinese hamster embryo cells transformed with SV40 [33]. In this case, the factor induced amplification of integrated SV40 sequences, a result that could involve production of acentric fragments [34]. T Antigen itself can also be considered a diffusible clastogenic factor; we recently showed that human diploid fibroblasts with an integrated SV40 A gene are mainly driven toward neoplastic transformation by ongoing clastogenicity and selection [35]. Although our data are consistent with a trans-acting factor, other possibilities have not been ruled ont. For instance, if the high cytogenetic instability of the CHX line is primarily a consequence of a heritably defective structure such as nuclear matrix or mitotic spindle [5], the CHX hybrids might have the same problem in maintaining stability of the chromosomes from either parent. The list of gene products that might have a role in chromosomal stability is very long indeed [4]; however, little direct evidence now exists to indicate which of them is(are) important in neoplasia. The consistent association of numerical and structural chromosomal changes in preneo-
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DIAGRAMS HUMAN-HAMSTER
OF
HYBRID
CHROMOSOMES
Ho-1
Hx7/1 A
A
B
B C
Hx5/3-7
B
D E F
C
G
D E F
H I J
G 0
