Chromosoma (Berl.) 62, 1- 15 (1977)
CHROMOSOMA 9 by Springer-Verlag 1977
Chromosomal Evolution in a Haploid Frog Cell Line: Implications for the Origin of Karyotypic Variants Liselotte Mezger-Freed The Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111, U.S.A.
Abstract. ICR 2A, a haploid cell line derived from Rana pipiens embryos, has remained haploid in number of chromosomes and their relative lengths and centromere positions for 500 cell generations. After this time, two new haryotypes appeared; relative length measurements indicate that the first has a translocation from chromosome 4 to 6, the second translocations from 3 and 4 to 6 and 7. The single exchange karyotype is not a precursor for the double exchange according to a statistical analysis. The double exchange karyotype characterized 90% of some cultures although a selective advantage could not be demonstrated for these cells. The observations suggest that a non-clonal or multicellular origin may account for these karyotypic variants. Introduction
The prediction ~that haploid cells would be an excellent source of cell culture mutants led to the establishment of the first stable haploid cell lines of vertebrate origin (Freed and Mezger-Freed, 1970a). Because these cell lines from the grass frog Rana pipiens have no counterpart from other vertebrate species, their chromosomal evolution is of some interest. Although 90-100% of the cells in line ICR 2A remained haploid in chromosome number and D N A content for over 500 cell generations (Mezger-Freed, 1977), a more detailed analysis of its karyotypic history was undertaken for several reasons. Aneuploidy is characteristic of most, perhaps all, animal cell lines derived from diploid tissues. The question of aneuploidy is of particular interest for the haploid cultures because the expected yield of mutants has not been realized and therefore the possibility of duplication of genetic material must be considered (Mezger-Freed, in press). The observations reported here indicate that karyotypic changes are restricted in haploid cells, perhaps because chromosome loss or imbalance would be less viable than in cells of higher ploidy. However, two new karyotypes present in unusually high proportions were observed; their history raises some questions about the origin of chromosomal markers in cell culture and malignancy.
2
L. Mezger-Freed
Materials and Methods The ICR 2A cell line was derived from haploid Rana pipiens embryos (Freed and Mezger-Freed,
1970a). (The designation "2AS50" identifies the 50th subculture, "2AS100" the 100th subculture, etc.) The cells are grown as a monolayer at 25~ in a modified Leibovitz medium (referred to as MS). Methods for culturing the amphibian cells are described in Freed and Mezger-Freed (1970b). The population doubling time is 50-60 h. The cell generation time or cell cycle transit time for ICR 2A is 37.8 + 13.5 h as determined by Dr. M.L. Mendelsohn (see Altman and Katz, 1976) using the computer method of Takahashi et al. (1971). Metaphase preparations were stained with Giemsa as described in Freed and Mezger-Freed (1970b). Metaphases were considered acceptable for analysis if all (26) of the chromosome arms could be distinguished in photographs taken with oil immersion optics. Arm lengths were then measured using a Numonics Graphics Calculator (Numonics Corporation, North WaIes, Pa.) and the results checked with a ruler. Relative chromosome lengths were calculated as the percent of the sum of all chromosome lengths in a metaphase; a centromere position is the length of the long arm/total length of the chromosome. The non-parametric Mann-Whitney test was used for the statistical analysis of chromosome lengths in consultation with Dr. Samuel Litwin. Determinations of the quantity of DNA per cell were by Dr. L.L. Deaven (Los Alamos) using a modification of the method of Kraemer et al. (1972).
Observations and Discussion
The Karyotype of the Haploid Cell Line ICR 2A If the c h r o m o s o m e s o f Rana pipiens are a r r a n g e d a c c o r d i n g to relative length, it is evident t h a t there are two classes, one with five large a n d the o t h e r with eight small c h r o m o s o m e s (Fig. 1). This d i c h o t o m y , c o m b i n e d with the small n u m b e r o f 13 c h r o m o s o m e s , m a k e s it possible to scan large n u m b e r s o f m e t a p h a s e plates for gross c h r o m o s o m a l changes as well as for the h a p l o i d c h r o m o some n u m b e r . F o r example, a scan o f 1505 cells (subcultures $69 to S138) i n d i c a t e d t h a t 99% o f I C R 2 A cells were h a p l o i d a c c o r d i n g to t o t a l c h r o m o s o m e n u m b e r a n d the presence o f 5 large c h r o m o s o m e s . A m o r e a c c u r a t e assessment o f the h a p l o i d o r e u p l o i d c o n d i t i o n o f I C R 2 A has been m a d e b y d e t e r m i n i n g relative c h r o m o s o m e lengths at various s u b c u l t u r e ages, using the c e n t r o m e r e p o s i t i o n s to help identify c h r o m o s o m e s (Table 1). The relative lengths o f chrom o s o m e s 4, I0, 11 a n d 13 in I C R 2 A S 3 2 were significantly different f r o m e m b r y o c h r o m o s o m e ( m e a s u r e d by D i B e r a r d i n o , 1962) a l t h o u g h the differences were each less t h a n 1% o f the t o t a l g e n o m e ( F r e e d a n d M e z g e r - F r e e d , 1970a) (Table 1, Fig. 1). W h e t h e r o r n o t a n y o f the a p p a r e n t s t r u c t u r a l changes are real, i.e., heritable, is q u e s t i o n a b l e , since at S l 1 6 a n d S l 1 7 these same f o u r c h r o m o s o m e s were f o u n d n o t to differ significantly f r o m those o f the e m b r y o s . C o m p a r i s o n s o f the s t a n d a r d d e v i a t i o n s o f c h r o m o s o m e lengths at S l 1 6 a n d S 117 indicate t h a t the k a r y o t y p e s o f the m e a s u r e d m e t a p h a s e s were as u n i f o r m in cultures as in e m b r y o s . The cultures o f I C R 2 A S l 1 6 h a d a b o u t 90% o f the m e t a p h a s e s s h o w i n g a h a p l o i d c h r o m o s o m e n u m b e r ; it h a d u n d e r g o n e a b o u t 530 cell g e n e r a t i o n s a c c o r d i n g to calculations using 40 h as the average length o f a cell generation. Thus, the results indicate t h a t an u n c l o n e d c o n t i n u o u s cell line can r e m a i n h a p l o i d in the sense o f h a v i n g a k a r y o t y p e with n o r m a l c h r o m o s o m e m o r p h o l o g y as well as the a p p r o p r i a t e n u m b e r o f c h r o m o s o m e s .
Chromosomes of a Haploid Frog Cell Line
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N
Embryo ICR $ 3 2
N
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ICR $116
.&E c(D J
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Chromosome
Fig. 1. Idiogram comparing metaphase chromosomes of Rana pipiens embryo with the haploid cell line ICR 2A at subcultures 32 and 1i6. The embryo data are from DiBerardino (1962). The relative lengths of individual chromosomes were determined in a total of 25 metaphases for each subculture, a total of 48 metaphases for the embryo
The Maintenance of Haploidy The earliest Rana pipiens cultures derived from haploid embryos became diploid shortly after the cell lines were initiated from haploid embryos (Freed, 1962). Although chromosome doubling has also been observed in diploid cell lines from a number of species, it is difficult to determine the part played by cell fusion, endomitotic duplication and selection in this process. Diploid cells have no apparent selective advantage since cultures of diploid cells do not multiply more rapidly than haploid cells. Whatever the cause of the increase in the proportion of diploid cells, cultures in medium containing the large molecular weight fraction of serum ( M P H medium, Sooy and Mezger-Freed, 1970) remain haploid longer than cultures in whole fetal bovine serum. In one experiment, cultures of I C R 2A remained 98-100% haploid for ten subcultures in M P H whereas in medium with full serum the proportion of haploid cells decreased from 100% to 4%. A cloned derivative of I C R 2A remained 80% haploid in the same M P H medium compared to 7% in MS medium. Since cultures multiply more slowly in M P H than in MS Medium, the cultures were handled so that the number of population doublings per subculture was similar in both media; therefore, the total number of cell generations does not account for
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L, Mezger-Freed
Table 1. Relative chromosome lengths and centromere positions from metaphases of the haploid
Rana pipiens cell line ICR 2A with the normal embryo measurements from DiBerardino (1962) shown for comparison. The centromere position for each chromosome is shown under the relative length. Standard deviations are in parenthesis. N=total number of metaphases measured. (H) identifies cultures that were maintained in MPH medium for at least one subculture before chromoCell Population
Chromosome number 1
2
3
4
5
Embryo N=48
15.4 (1.0) 0.53
12.4 (0.9) 0.66
12.1 (0.8) 0.72
11.5 (0.6) 0.55
9.5 (0.7) 0.53
2AS32 N=25
15.5 (0.8) 0.55 (0.01)
12.4 (0.4) 0.66 (0.01)
12.3 (0.6) 0.72 (0.01)
12.2 (0.7) 0.57 (0.1)
9.3 (0.5) 0.53 (0.01)
2ASl16 N=25
15.3 (1.0) 0.56 (0.03)
12.4 (0.9) 0.67 (0.03)
11.8 (0.7) 0.73 (0.02)
11.7 (0.7) 0.58 (0.03)
9.4 (0.8) 0.54 (0.02)
2A(H)Sll7 N = 19
15.0 (1.0) 0.54 (0.02)
12.0 (0.7) 0.66 (0.03)
12.2 (0.8) 0.73 (0.02)
11.4 (0.8) 0.57 (0.02)
9.2 (0.6) 0.55 (0.03)
2A(H)S120 Normal N = 19 Single X N = 10 Double X N = 19
14.9 0.56 14.4 0.55 15.2 0.55
(0.6) (0.04) (0.9) (0.02) (0.8) (0.02)
12.1 (0.6) 0.66 (0.02) 12.3 (0.8) 0.65 (0.02) 12.0 (0.6) 0.64 (0.01)
11.6 (0.8) 0.72 (0.03) 12.7 (0.8) 0.72 (0.02) 10.1 (0.9) 0.66 (0.04)
11.5 (0.7) 0.59 (0.04) 10.0 (0.4) 0.73 (0.03) 9.5 (0.5) 0.66 (0.04)
9.5 0.54 9.3 0.55 9.0 0.54
2A(H)S126 N = 18
15.1 (0.8) 0.55 (0.02)
12.1 (0.8) 0.65 (0.02)
10.0 (0.6) 0.67 (0.02)
9.2 (0.6) 0.66 (0.03)
9.3 (0.6) 0.54 (0.02)
2AS127 N=25
14.7 (0.9) 0.55 (0.02)
11.9 (0.5) 0.66 (0.02)
9.8 (0.5) 0.66 (0.04)
9.1 (0.4) 0.66 (0.03)
9.3 (0.6) 0.53 (0.02)
2A(H)S129 N=25
14.8 (1.0) 0.55 (0.02)
12.8 (0.8) 0.66 (0.03)
10.1 (0.7) 0.68 (0.04)
9.3 (0.5) 0.67 (0.04)
9.3 (0.7) 0.54 (0.03)
2AS132-134 N=25
15.3 (0.6) 0.55 (0.02)
12.3 (0.8) 0.66 (0.02)
lO.O (0.5) 0.67 (0.01)
9.2 (0.3) 0.66 (0.02)
9.4 (0.7) 0.53 (0.02)
2AS175 N=25
15.8 (0.9) 0.55 (0.02)
12.5 (0.8) 0.67 (0.03)
12.5 (0.6) 0.73 (0.03)
9.8 (0.6) 0.76 (0.04)
9.5 (0.7) 0.54 (0.03)
(0.8) (0.03) (0.4) (0.04) (0.6) (0.02)
the difference. It s h o u l d be n o t e d t h a t the cell line I C R 2 A r e m a i n e d h a p l o i d for 500 cell g e n e r a t i o n s in m e d i u m with w h o l e s e r u m a n d t h e r e f o r e the p r e s e n c e o f w h o l e s e r u m does n o t by itself affect c h a n g e s in the p l o i d y c o m p o s i t i o n o f cultures.
New Karyotypes in the ICR 2A Cell Line Metaphases five " l a r g e " h a d b e e n in was e v i d e n t
w i t h the h a p l o i d c h r o m o s o m e n u m b e r b u t w i t h seven i n s t e a d o f c h r o m o s o m e s were d i s c o v e r e d in c u l t u r e s o f I C R 2 A ( H ) S 126 t h a t M P H m e d i u m ( T a b l e 1, Figs. 2 a n d 3). A t this stage the a l t e r a t i o n b e c a u s e at least 8 0 % o f the p o p u l a t i o n c o n s i s t e d o f the n e w k a r y o -
Chromosomes of a Haploid Frog Cell Line
5
some preparations were made; otherwise MS medium was used. Single X refers to karyotypes in which a translocation between chromosome//4 and r has occurred ; double X refers to karyotypes with translocations from//3 to #6 or #7 and, in addition #4 to #6 or //7. "New" chromosomes are in the enclosed portions of the table
6
7
8
9
10
i1
12
13
6.0 (0.6) 0.65
6.2 (0.7) 0.54
4.9 (0.4) 0.64
4.4 (0.5) 0.65
4.8 (0.5) 0.54
4.5 (0.6) 0.68
4.0 (0.5) 0.69
4.3 (0.4) 0.55
6.1 (0.6) 0.64 (0.01)
6.0 (0.4) 0.53 (0.01)
5.1 (0.3) 0.65 (0.01)
4.7 (0.3) 0.64 (0.01)
4.4 (0.4) 0.57 (0.0I)
4.1 (0.3) 0.65 (0.01)
4.0 (0.3) 0.63 (0.01)
3.9 (0.3) 0.56 (0.01)
6.0 (0.5) 0.62 (0.03)
5.8 (0.5) 0.53 (0.03)
5.0 (0.4) 0.65 (0.04)
4.9 (0.5) 0.61 (0.06)
4.9 (0.4) 0.55 (0.03)
4.3 (0.5) 0.64 (0.05)
4.3 (0.5) 0.62 (0.05)
4.5 (0.4)
6.1 (0.4) 0.62 (0.04)
6.0 (0.5) 0.54 (0.03)
5.3 (0.4) 0.62 (0.03)
4.9 (0.4) 0.62 (0.06)
4.9 (0.3) 0.54 (0.04)
4.5 (0.4) 0.62 (0.03)
4.2 (0.3) 0.62 (0.03)
4.4 (0.3) 0.56 (0.04)
6.2 (0.5) 0.64 (0.06) 7.1 (0.4) 0.73 (0.02) 8.4 (0.5) 0.76 (0.04)
5.8 (0.4) 0.55 (0.03) 6.i (0.3) 0.57 (0.06) 8.0 (0.5) 0.72 (0.03)
5.4 (0.3) 0.64 (0.03) 5.6 (0.5) 0.64 (0.04) 5.0 (0.4) 0.64 (0.04)
5.0 (0.3) 0.62 (0,03) 5.0 (0.4) 0.64 (0.03) 4.8 (0.3) 0.59 (0.05)
5.0 (0.3) 0.56 (0.04) 4.7 (0.5) 0.56 (0.04) 5.0 (0.5) 0.53 (0.02)
4.4 (0.4) 0.62 (0.04) 4.6 (0.4) 0.64 (0.03) 4.1 (0.3) 0.63 (0.05)
4.5 (0.3) 0.60 (0.04) 4.3 (0.2) 0.61 (0.04) 4.3 (0.4) 0.59 (0.03)
4.3 (0.6) 0.56 (0.04) 4.0 (0.4) 0.56 (0.03) 4.6 (0.3) 0.54 (0.04)
8.4 (0.5) 0.75 (0.05)
7.8 (0.5) 0.71 (0.06)
5,4 (0.4) 0.59 (0.06)
5.1 (0,3) 0.58 (0.06)
4.8 (0.5) 0.45 (0.03)
4.1 (0.7) 0.63 (0.06)
4.5 (0.4) 0.60 (0.04)
4.1 (0.5) 0.57 (0.05)
8.4 (0.3) 0.75 (0.03)
7,8 (0.7) [ 5.4 (0.5) 0.70 (0.05) I 0.59 (0.03)
5.0 (0.3) 0.58 (0.04)
5.2 (0.3) 0.53 (0.03)
4.4 (0.4) 0.59 (0.04)
4.5 (0.4) 0.60 (0.03)
4.6 (0.5) 0.56 (0.04)
8.4 (0.4) 0.74 (0.05)
7.8 (0.5)[ 5.4 (0.4) 0.71 (0.05) [ 0.58 (0.05) I 7.9 (0.4) 5.4 (0.5) 0.71 (0.02) 0.59 (0.04) 6.0 (0.6) 5.9 (0.4) 0.54 (0.03) 0.65 (0.04)
4.8 (0.5) 0.61 (0.05)
5.0 (0.4) 0.53 (0.04)
4.0 (0.6) 0.60 (0.04)
4.3 (0.6) 0.60 (0.05)
4.2 (0.3) 0.55 (0.04)
4.7 (0.4) 0.59 (0.05)
4.8 (0.5) 0.53 (0.03)
3.9 (0.6) 0.58 (0.04)
4.3 (0.4) 0.59 (0.04)
4.3 (0.4) 0.56 (0.05)
4.9 (0.4) 0.64 (0.04)
4.6 (0.4) 0.58 (0.03)
3.8 (0.4) 0.61 (0.05)
4.1 (0.5) 0.62 (0.05)
3.8 (0.4) 0.56 (0.03)
8.5 (0.5) 0.77 (0.02) 6.9 (0.5) 0.72 (0.03)
0.55 (0.03)
t y p e w h e r e a s p r e v i o u s I C R 2 A c u l t u r e s e x a m i n e d h a d been largely n o r m a l . It w a s p o s s i b l e to r e c o n s t r u c t m u c h o f the h i s t o r y o f these c h r o m o s o m a l c h a n g e s from stained preparations and from frozen samples of previous subcultures ( T a b l e 2). T h e D N A c o n t e n t ( g e n o m e ) o f G1 cells f r o m the n e w p o p u l a t i o n was i n d i s t i n g u i s h a b l e f r o m n o r m a l b y flow m i c r o f l u o r i m e t r y ( c o u r t e s y o f Dr. L.L. D e a v e n ) thus m a k i n g it v a l i d to c o m p a r e relative lengths o f c h r o m o s o m e s f r o m the t w o types. F o u r o f the c h r o m o s o m e s f r o m the n o r m a l k a r y o t y p e , //3, 4, 6, a n d 7 were r e p l a c e d b y f o u r " n e w " c h r o m o s o m e s t h a t c o u l d h a v e a r i s e n b y the t r a n s f e r o f m a t e r i a l f r o m the l o n g a r m (q) o f / / 3 a n d the s h o r t a r m (p) o f / / 4 t o / / 6 q a n d H7p o r //7% (Fig. 2) T h e t r a n s f e r r e d p o r t i o n s e a c h c o n s t i t u t e a b o u t 2 % o f the t o t a l g e n o m e length, too s m a l l to p u t //3 a n d //4 in the " s h o r t " class b u t large e n o u g h so the eye d i s c e r n s the new //6 a n d
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L. Mezger-Freed 15
10
t-
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6 7 8 Chromosome
9
10
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12
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15
O3 C
10 d
rr
15' ,,\
%\
10-
1
2
3
4
5
Fig. 2. Idiograms representing chromosome lengths of normal metaphases (5 large chromosomes), single exchange metaphases (6 large chromosomes) and double exchange metaphases (7 large chromosomes) of ICR 2A(H)S120. In the idiograms, dark areas represent the relative chromosome lengths with the relative lengths of the chromosome arms in squares. Stippled areas are the portions translocated from the normal chromosomes, hatched areas the portions translocated to the normal chromosomes according to the hypothesis presented in the text. Note that in the double exchange the measurements c a n n o t identify whether the translocations are between //3 and //6 (//4 and #7) or between #3 and//7 (//4 and//6)
Chromosomes of a Haploid Frog Cell Line
7
~g
Ca, o
E 3~
fi
?~..~
r--
.~
//7 as "large chromosomes". The break in //3q occurred near a constriction observed by DiBerardino (1962) in embryo chromosomes. The new "double exchange" phases constitute a uniform karyotype; the coefficient of variation for the four new chromosomes is not greater than for normal chromosomes. The new karyotype could be detected as early as subculture 120 after three passages in MPH medium; in a total of 50 metaphases 19 (38%) were of the normal karyotype, an equal number were double exchange (Table 2). In
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L. Mezger-Freed
Table 2. Karyotype analysis of I C R 2A populations with double exchange chromosomes Subcnlture
Sl16 (H)SLI7 (H)S120 (H)S124 a (H)S126 (H)S126"
Normal
Double exchange
Single exchange
Other
No.
%
No.
%
No.
%
No.
%
24 19 19 1 1 0
92 83 38 3 3 0
0 0 19 30 25 98
0 0 38 b 81 83 98
1 1 10 6 1 2
4 4 20 16 3 2
1 3 2 0 3 0
4 13 4 0 i0 0
a Karyotypes were classified according to the relative lengths of chromosomes (see text) unless marked with an a; in that case the n u m b e r of large chromosomes per metaphase was determined (7 large = double exchange; 6 large = single exchange) b The n u m b e r of population doublings (D) required for a single variant cell to reach a proportion of the population equal to $, when p equals the initial proportion and p equals the difference in growth rate, can be obtained by computer analysis using the formula p 2 v/(l= v) 9 ~ 2 o -- p 2 D/tl- p) Thus, if a single cell with the double exchange karyotype arose in a population of 5 x 104 cells, it would require 60 doublings to reach the 38% proportion at (H) S120 with a growth rate advantage of 20%, 35 doublings with an advantage of 30% and 15 doublings with an advantage of 50%
addition a third karyotype was present in 20% of the population; it differed from normal in having a translocation from //4p to #6q. An example of this karyotype was also found in ICR 2ASl16 and in S(H)ll7 (Table 2). The nonparametric Mann-Whitney test was applied in order to test whether the single exchange could be an intermediate for the double exchange as well as to establish whether the new karyotype classes were significantly different from the normal ICR 2A karyotype. The analysis in Table 3 was made on metaphases from a single culture, thus decreasing the possibility of artifacts from staining and culture procedures. The double exchange karyotype, involving c h r o m o s o m e s 3, 4, 6, and 7, can be identified by the presence of a //7 chromosome with relative length 8.0% and centromere position 0.72. If the karyotypes at S120 are divided into two classes, one with #7q longer than 5.0% (double exchange) and the other shorter than 4.0% (normal and single exchange), these classes show highly significant differences in the #3 chromosome ( P < 10-5) whereas, for example, the difference in /41 is not significant (P=0.24). Thus, ICR 2A(H)S120 metaphases contained two significantly different classes of//3 chromosomes, the shorter of which was associated with the longer//7. If the double exchange karyotype is classified by r and the single exchange distinguished from normal by the lengths and centromere positions o f / / 4 and ~/6, then a statistical analysis shows that the differences postulated for the four chromosome arms are significant (Table 3). Furthermore, the analysis indicates that //6 is not the same length in the two new karyotypes; thus it is unlikely that the single exchange was a precursor of the double exchange. Whether there has
C h r o m o s o m e s of a Haploid Frog Cell Line Table 3. Analysis of differences in relative lengths of c h r o m o s o m e arms in I C R 2A(H)S120 Chromosome
3q 4p 6q 7q
Karyotype %
Differences
Normal (S.D.)
Single Double exchange exchange (S.D.) (S.D.)
Normal vs. single exchange
Normal vs. double exchange
Single vs. double exchange
8.3(0.6) 4.7(0.6) 4.0(0.6) 3.1 (0.3)
9.1(0.8) 2.6(0.3) 5.2(0.3) 3.4(0.3)
+0.8 -2.1 +1.2 +0.3
--1.6 -1.6 +2.5 +2.7
2.4 0.5 1.3 2.4
6.7(0.5) 3.1(0.6) 6.5(0.7) 5.8(0.5)
P=10 -2 P
CHROMOSOMA 9 by Springer-Verlag 1977
Chromosomal Evolution in a Haploid Frog Cell Line: Implications for the Origin of Karyotypic Variants Liselotte Mezger-Freed The Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111, U.S.A.
Abstract. ICR 2A, a haploid cell line derived from Rana pipiens embryos, has remained haploid in number of chromosomes and their relative lengths and centromere positions for 500 cell generations. After this time, two new haryotypes appeared; relative length measurements indicate that the first has a translocation from chromosome 4 to 6, the second translocations from 3 and 4 to 6 and 7. The single exchange karyotype is not a precursor for the double exchange according to a statistical analysis. The double exchange karyotype characterized 90% of some cultures although a selective advantage could not be demonstrated for these cells. The observations suggest that a non-clonal or multicellular origin may account for these karyotypic variants. Introduction
The prediction ~that haploid cells would be an excellent source of cell culture mutants led to the establishment of the first stable haploid cell lines of vertebrate origin (Freed and Mezger-Freed, 1970a). Because these cell lines from the grass frog Rana pipiens have no counterpart from other vertebrate species, their chromosomal evolution is of some interest. Although 90-100% of the cells in line ICR 2A remained haploid in chromosome number and D N A content for over 500 cell generations (Mezger-Freed, 1977), a more detailed analysis of its karyotypic history was undertaken for several reasons. Aneuploidy is characteristic of most, perhaps all, animal cell lines derived from diploid tissues. The question of aneuploidy is of particular interest for the haploid cultures because the expected yield of mutants has not been realized and therefore the possibility of duplication of genetic material must be considered (Mezger-Freed, in press). The observations reported here indicate that karyotypic changes are restricted in haploid cells, perhaps because chromosome loss or imbalance would be less viable than in cells of higher ploidy. However, two new karyotypes present in unusually high proportions were observed; their history raises some questions about the origin of chromosomal markers in cell culture and malignancy.
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L. Mezger-Freed
Materials and Methods The ICR 2A cell line was derived from haploid Rana pipiens embryos (Freed and Mezger-Freed,
1970a). (The designation "2AS50" identifies the 50th subculture, "2AS100" the 100th subculture, etc.) The cells are grown as a monolayer at 25~ in a modified Leibovitz medium (referred to as MS). Methods for culturing the amphibian cells are described in Freed and Mezger-Freed (1970b). The population doubling time is 50-60 h. The cell generation time or cell cycle transit time for ICR 2A is 37.8 + 13.5 h as determined by Dr. M.L. Mendelsohn (see Altman and Katz, 1976) using the computer method of Takahashi et al. (1971). Metaphase preparations were stained with Giemsa as described in Freed and Mezger-Freed (1970b). Metaphases were considered acceptable for analysis if all (26) of the chromosome arms could be distinguished in photographs taken with oil immersion optics. Arm lengths were then measured using a Numonics Graphics Calculator (Numonics Corporation, North WaIes, Pa.) and the results checked with a ruler. Relative chromosome lengths were calculated as the percent of the sum of all chromosome lengths in a metaphase; a centromere position is the length of the long arm/total length of the chromosome. The non-parametric Mann-Whitney test was used for the statistical analysis of chromosome lengths in consultation with Dr. Samuel Litwin. Determinations of the quantity of DNA per cell were by Dr. L.L. Deaven (Los Alamos) using a modification of the method of Kraemer et al. (1972).
Observations and Discussion
The Karyotype of the Haploid Cell Line ICR 2A If the c h r o m o s o m e s o f Rana pipiens are a r r a n g e d a c c o r d i n g to relative length, it is evident t h a t there are two classes, one with five large a n d the o t h e r with eight small c h r o m o s o m e s (Fig. 1). This d i c h o t o m y , c o m b i n e d with the small n u m b e r o f 13 c h r o m o s o m e s , m a k e s it possible to scan large n u m b e r s o f m e t a p h a s e plates for gross c h r o m o s o m a l changes as well as for the h a p l o i d c h r o m o some n u m b e r . F o r example, a scan o f 1505 cells (subcultures $69 to S138) i n d i c a t e d t h a t 99% o f I C R 2 A cells were h a p l o i d a c c o r d i n g to t o t a l c h r o m o s o m e n u m b e r a n d the presence o f 5 large c h r o m o s o m e s . A m o r e a c c u r a t e assessment o f the h a p l o i d o r e u p l o i d c o n d i t i o n o f I C R 2 A has been m a d e b y d e t e r m i n i n g relative c h r o m o s o m e lengths at various s u b c u l t u r e ages, using the c e n t r o m e r e p o s i t i o n s to help identify c h r o m o s o m e s (Table 1). The relative lengths o f chrom o s o m e s 4, I0, 11 a n d 13 in I C R 2 A S 3 2 were significantly different f r o m e m b r y o c h r o m o s o m e ( m e a s u r e d by D i B e r a r d i n o , 1962) a l t h o u g h the differences were each less t h a n 1% o f the t o t a l g e n o m e ( F r e e d a n d M e z g e r - F r e e d , 1970a) (Table 1, Fig. 1). W h e t h e r o r n o t a n y o f the a p p a r e n t s t r u c t u r a l changes are real, i.e., heritable, is q u e s t i o n a b l e , since at S l 1 6 a n d S l 1 7 these same f o u r c h r o m o s o m e s were f o u n d n o t to differ significantly f r o m those o f the e m b r y o s . C o m p a r i s o n s o f the s t a n d a r d d e v i a t i o n s o f c h r o m o s o m e lengths at S l 1 6 a n d S 117 indicate t h a t the k a r y o t y p e s o f the m e a s u r e d m e t a p h a s e s were as u n i f o r m in cultures as in e m b r y o s . The cultures o f I C R 2 A S l 1 6 h a d a b o u t 90% o f the m e t a p h a s e s s h o w i n g a h a p l o i d c h r o m o s o m e n u m b e r ; it h a d u n d e r g o n e a b o u t 530 cell g e n e r a t i o n s a c c o r d i n g to calculations using 40 h as the average length o f a cell generation. Thus, the results indicate t h a t an u n c l o n e d c o n t i n u o u s cell line can r e m a i n h a p l o i d in the sense o f h a v i n g a k a r y o t y p e with n o r m a l c h r o m o s o m e m o r p h o l o g y as well as the a p p r o p r i a t e n u m b e r o f c h r o m o s o m e s .
Chromosomes of a Haploid Frog Cell Line
3
15
N
Embryo ICR $ 3 2
N
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ICR $116
.&E c(D J
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Chromosome
Fig. 1. Idiogram comparing metaphase chromosomes of Rana pipiens embryo with the haploid cell line ICR 2A at subcultures 32 and 1i6. The embryo data are from DiBerardino (1962). The relative lengths of individual chromosomes were determined in a total of 25 metaphases for each subculture, a total of 48 metaphases for the embryo
The Maintenance of Haploidy The earliest Rana pipiens cultures derived from haploid embryos became diploid shortly after the cell lines were initiated from haploid embryos (Freed, 1962). Although chromosome doubling has also been observed in diploid cell lines from a number of species, it is difficult to determine the part played by cell fusion, endomitotic duplication and selection in this process. Diploid cells have no apparent selective advantage since cultures of diploid cells do not multiply more rapidly than haploid cells. Whatever the cause of the increase in the proportion of diploid cells, cultures in medium containing the large molecular weight fraction of serum ( M P H medium, Sooy and Mezger-Freed, 1970) remain haploid longer than cultures in whole fetal bovine serum. In one experiment, cultures of I C R 2A remained 98-100% haploid for ten subcultures in M P H whereas in medium with full serum the proportion of haploid cells decreased from 100% to 4%. A cloned derivative of I C R 2A remained 80% haploid in the same M P H medium compared to 7% in MS medium. Since cultures multiply more slowly in M P H than in MS Medium, the cultures were handled so that the number of population doublings per subculture was similar in both media; therefore, the total number of cell generations does not account for
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Table 1. Relative chromosome lengths and centromere positions from metaphases of the haploid
Rana pipiens cell line ICR 2A with the normal embryo measurements from DiBerardino (1962) shown for comparison. The centromere position for each chromosome is shown under the relative length. Standard deviations are in parenthesis. N=total number of metaphases measured. (H) identifies cultures that were maintained in MPH medium for at least one subculture before chromoCell Population
Chromosome number 1
2
3
4
5
Embryo N=48
15.4 (1.0) 0.53
12.4 (0.9) 0.66
12.1 (0.8) 0.72
11.5 (0.6) 0.55
9.5 (0.7) 0.53
2AS32 N=25
15.5 (0.8) 0.55 (0.01)
12.4 (0.4) 0.66 (0.01)
12.3 (0.6) 0.72 (0.01)
12.2 (0.7) 0.57 (0.1)
9.3 (0.5) 0.53 (0.01)
2ASl16 N=25
15.3 (1.0) 0.56 (0.03)
12.4 (0.9) 0.67 (0.03)
11.8 (0.7) 0.73 (0.02)
11.7 (0.7) 0.58 (0.03)
9.4 (0.8) 0.54 (0.02)
2A(H)Sll7 N = 19
15.0 (1.0) 0.54 (0.02)
12.0 (0.7) 0.66 (0.03)
12.2 (0.8) 0.73 (0.02)
11.4 (0.8) 0.57 (0.02)
9.2 (0.6) 0.55 (0.03)
2A(H)S120 Normal N = 19 Single X N = 10 Double X N = 19
14.9 0.56 14.4 0.55 15.2 0.55
(0.6) (0.04) (0.9) (0.02) (0.8) (0.02)
12.1 (0.6) 0.66 (0.02) 12.3 (0.8) 0.65 (0.02) 12.0 (0.6) 0.64 (0.01)
11.6 (0.8) 0.72 (0.03) 12.7 (0.8) 0.72 (0.02) 10.1 (0.9) 0.66 (0.04)
11.5 (0.7) 0.59 (0.04) 10.0 (0.4) 0.73 (0.03) 9.5 (0.5) 0.66 (0.04)
9.5 0.54 9.3 0.55 9.0 0.54
2A(H)S126 N = 18
15.1 (0.8) 0.55 (0.02)
12.1 (0.8) 0.65 (0.02)
10.0 (0.6) 0.67 (0.02)
9.2 (0.6) 0.66 (0.03)
9.3 (0.6) 0.54 (0.02)
2AS127 N=25
14.7 (0.9) 0.55 (0.02)
11.9 (0.5) 0.66 (0.02)
9.8 (0.5) 0.66 (0.04)
9.1 (0.4) 0.66 (0.03)
9.3 (0.6) 0.53 (0.02)
2A(H)S129 N=25
14.8 (1.0) 0.55 (0.02)
12.8 (0.8) 0.66 (0.03)
10.1 (0.7) 0.68 (0.04)
9.3 (0.5) 0.67 (0.04)
9.3 (0.7) 0.54 (0.03)
2AS132-134 N=25
15.3 (0.6) 0.55 (0.02)
12.3 (0.8) 0.66 (0.02)
lO.O (0.5) 0.67 (0.01)
9.2 (0.3) 0.66 (0.02)
9.4 (0.7) 0.53 (0.02)
2AS175 N=25
15.8 (0.9) 0.55 (0.02)
12.5 (0.8) 0.67 (0.03)
12.5 (0.6) 0.73 (0.03)
9.8 (0.6) 0.76 (0.04)
9.5 (0.7) 0.54 (0.03)
(0.8) (0.03) (0.4) (0.04) (0.6) (0.02)
the difference. It s h o u l d be n o t e d t h a t the cell line I C R 2 A r e m a i n e d h a p l o i d for 500 cell g e n e r a t i o n s in m e d i u m with w h o l e s e r u m a n d t h e r e f o r e the p r e s e n c e o f w h o l e s e r u m does n o t by itself affect c h a n g e s in the p l o i d y c o m p o s i t i o n o f cultures.
New Karyotypes in the ICR 2A Cell Line Metaphases five " l a r g e " h a d b e e n in was e v i d e n t
w i t h the h a p l o i d c h r o m o s o m e n u m b e r b u t w i t h seven i n s t e a d o f c h r o m o s o m e s were d i s c o v e r e d in c u l t u r e s o f I C R 2 A ( H ) S 126 t h a t M P H m e d i u m ( T a b l e 1, Figs. 2 a n d 3). A t this stage the a l t e r a t i o n b e c a u s e at least 8 0 % o f the p o p u l a t i o n c o n s i s t e d o f the n e w k a r y o -
Chromosomes of a Haploid Frog Cell Line
5
some preparations were made; otherwise MS medium was used. Single X refers to karyotypes in which a translocation between chromosome//4 and r has occurred ; double X refers to karyotypes with translocations from//3 to #6 or #7 and, in addition #4 to #6 or //7. "New" chromosomes are in the enclosed portions of the table
6
7
8
9
10
i1
12
13
6.0 (0.6) 0.65
6.2 (0.7) 0.54
4.9 (0.4) 0.64
4.4 (0.5) 0.65
4.8 (0.5) 0.54
4.5 (0.6) 0.68
4.0 (0.5) 0.69
4.3 (0.4) 0.55
6.1 (0.6) 0.64 (0.01)
6.0 (0.4) 0.53 (0.01)
5.1 (0.3) 0.65 (0.01)
4.7 (0.3) 0.64 (0.01)
4.4 (0.4) 0.57 (0.0I)
4.1 (0.3) 0.65 (0.01)
4.0 (0.3) 0.63 (0.01)
3.9 (0.3) 0.56 (0.01)
6.0 (0.5) 0.62 (0.03)
5.8 (0.5) 0.53 (0.03)
5.0 (0.4) 0.65 (0.04)
4.9 (0.5) 0.61 (0.06)
4.9 (0.4) 0.55 (0.03)
4.3 (0.5) 0.64 (0.05)
4.3 (0.5) 0.62 (0.05)
4.5 (0.4)
6.1 (0.4) 0.62 (0.04)
6.0 (0.5) 0.54 (0.03)
5.3 (0.4) 0.62 (0.03)
4.9 (0.4) 0.62 (0.06)
4.9 (0.3) 0.54 (0.04)
4.5 (0.4) 0.62 (0.03)
4.2 (0.3) 0.62 (0.03)
4.4 (0.3) 0.56 (0.04)
6.2 (0.5) 0.64 (0.06) 7.1 (0.4) 0.73 (0.02) 8.4 (0.5) 0.76 (0.04)
5.8 (0.4) 0.55 (0.03) 6.i (0.3) 0.57 (0.06) 8.0 (0.5) 0.72 (0.03)
5.4 (0.3) 0.64 (0.03) 5.6 (0.5) 0.64 (0.04) 5.0 (0.4) 0.64 (0.04)
5.0 (0.3) 0.62 (0,03) 5.0 (0.4) 0.64 (0.03) 4.8 (0.3) 0.59 (0.05)
5.0 (0.3) 0.56 (0.04) 4.7 (0.5) 0.56 (0.04) 5.0 (0.5) 0.53 (0.02)
4.4 (0.4) 0.62 (0.04) 4.6 (0.4) 0.64 (0.03) 4.1 (0.3) 0.63 (0.05)
4.5 (0.3) 0.60 (0.04) 4.3 (0.2) 0.61 (0.04) 4.3 (0.4) 0.59 (0.03)
4.3 (0.6) 0.56 (0.04) 4.0 (0.4) 0.56 (0.03) 4.6 (0.3) 0.54 (0.04)
8.4 (0.5) 0.75 (0.05)
7.8 (0.5) 0.71 (0.06)
5,4 (0.4) 0.59 (0.06)
5.1 (0,3) 0.58 (0.06)
4.8 (0.5) 0.45 (0.03)
4.1 (0.7) 0.63 (0.06)
4.5 (0.4) 0.60 (0.04)
4.1 (0.5) 0.57 (0.05)
8.4 (0.3) 0.75 (0.03)
7,8 (0.7) [ 5.4 (0.5) 0.70 (0.05) I 0.59 (0.03)
5.0 (0.3) 0.58 (0.04)
5.2 (0.3) 0.53 (0.03)
4.4 (0.4) 0.59 (0.04)
4.5 (0.4) 0.60 (0.03)
4.6 (0.5) 0.56 (0.04)
8.4 (0.4) 0.74 (0.05)
7.8 (0.5)[ 5.4 (0.4) 0.71 (0.05) [ 0.58 (0.05) I 7.9 (0.4) 5.4 (0.5) 0.71 (0.02) 0.59 (0.04) 6.0 (0.6) 5.9 (0.4) 0.54 (0.03) 0.65 (0.04)
4.8 (0.5) 0.61 (0.05)
5.0 (0.4) 0.53 (0.04)
4.0 (0.6) 0.60 (0.04)
4.3 (0.6) 0.60 (0.05)
4.2 (0.3) 0.55 (0.04)
4.7 (0.4) 0.59 (0.05)
4.8 (0.5) 0.53 (0.03)
3.9 (0.6) 0.58 (0.04)
4.3 (0.4) 0.59 (0.04)
4.3 (0.4) 0.56 (0.05)
4.9 (0.4) 0.64 (0.04)
4.6 (0.4) 0.58 (0.03)
3.8 (0.4) 0.61 (0.05)
4.1 (0.5) 0.62 (0.05)
3.8 (0.4) 0.56 (0.03)
8.5 (0.5) 0.77 (0.02) 6.9 (0.5) 0.72 (0.03)
0.55 (0.03)
t y p e w h e r e a s p r e v i o u s I C R 2 A c u l t u r e s e x a m i n e d h a d been largely n o r m a l . It w a s p o s s i b l e to r e c o n s t r u c t m u c h o f the h i s t o r y o f these c h r o m o s o m a l c h a n g e s from stained preparations and from frozen samples of previous subcultures ( T a b l e 2). T h e D N A c o n t e n t ( g e n o m e ) o f G1 cells f r o m the n e w p o p u l a t i o n was i n d i s t i n g u i s h a b l e f r o m n o r m a l b y flow m i c r o f l u o r i m e t r y ( c o u r t e s y o f Dr. L.L. D e a v e n ) thus m a k i n g it v a l i d to c o m p a r e relative lengths o f c h r o m o s o m e s f r o m the t w o types. F o u r o f the c h r o m o s o m e s f r o m the n o r m a l k a r y o t y p e , //3, 4, 6, a n d 7 were r e p l a c e d b y f o u r " n e w " c h r o m o s o m e s t h a t c o u l d h a v e a r i s e n b y the t r a n s f e r o f m a t e r i a l f r o m the l o n g a r m (q) o f / / 3 a n d the s h o r t a r m (p) o f / / 4 t o / / 6 q a n d H7p o r //7% (Fig. 2) T h e t r a n s f e r r e d p o r t i o n s e a c h c o n s t i t u t e a b o u t 2 % o f the t o t a l g e n o m e length, too s m a l l to p u t //3 a n d //4 in the " s h o r t " class b u t large e n o u g h so the eye d i s c e r n s the new //6 a n d
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L. Mezger-Freed 15
10
t-
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6 7 8 Chromosome
9
10
11
12
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15
O3 C
10 d
rr
15' ,,\
%\
10-
1
2
3
4
5
Fig. 2. Idiograms representing chromosome lengths of normal metaphases (5 large chromosomes), single exchange metaphases (6 large chromosomes) and double exchange metaphases (7 large chromosomes) of ICR 2A(H)S120. In the idiograms, dark areas represent the relative chromosome lengths with the relative lengths of the chromosome arms in squares. Stippled areas are the portions translocated from the normal chromosomes, hatched areas the portions translocated to the normal chromosomes according to the hypothesis presented in the text. Note that in the double exchange the measurements c a n n o t identify whether the translocations are between //3 and //6 (//4 and #7) or between #3 and//7 (//4 and//6)
Chromosomes of a Haploid Frog Cell Line
7
~g
Ca, o
E 3~
fi
?~..~
r--
.~
//7 as "large chromosomes". The break in //3q occurred near a constriction observed by DiBerardino (1962) in embryo chromosomes. The new "double exchange" phases constitute a uniform karyotype; the coefficient of variation for the four new chromosomes is not greater than for normal chromosomes. The new karyotype could be detected as early as subculture 120 after three passages in MPH medium; in a total of 50 metaphases 19 (38%) were of the normal karyotype, an equal number were double exchange (Table 2). In
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Table 2. Karyotype analysis of I C R 2A populations with double exchange chromosomes Subcnlture
Sl16 (H)SLI7 (H)S120 (H)S124 a (H)S126 (H)S126"
Normal
Double exchange
Single exchange
Other
No.
%
No.
%
No.
%
No.
%
24 19 19 1 1 0
92 83 38 3 3 0
0 0 19 30 25 98
0 0 38 b 81 83 98
1 1 10 6 1 2
4 4 20 16 3 2
1 3 2 0 3 0
4 13 4 0 i0 0
a Karyotypes were classified according to the relative lengths of chromosomes (see text) unless marked with an a; in that case the n u m b e r of large chromosomes per metaphase was determined (7 large = double exchange; 6 large = single exchange) b The n u m b e r of population doublings (D) required for a single variant cell to reach a proportion of the population equal to $, when p equals the initial proportion and p equals the difference in growth rate, can be obtained by computer analysis using the formula p 2 v/(l= v) 9 ~ 2 o -- p 2 D/tl- p) Thus, if a single cell with the double exchange karyotype arose in a population of 5 x 104 cells, it would require 60 doublings to reach the 38% proportion at (H) S120 with a growth rate advantage of 20%, 35 doublings with an advantage of 30% and 15 doublings with an advantage of 50%
addition a third karyotype was present in 20% of the population; it differed from normal in having a translocation from //4p to #6q. An example of this karyotype was also found in ICR 2ASl16 and in S(H)ll7 (Table 2). The nonparametric Mann-Whitney test was applied in order to test whether the single exchange could be an intermediate for the double exchange as well as to establish whether the new karyotype classes were significantly different from the normal ICR 2A karyotype. The analysis in Table 3 was made on metaphases from a single culture, thus decreasing the possibility of artifacts from staining and culture procedures. The double exchange karyotype, involving c h r o m o s o m e s 3, 4, 6, and 7, can be identified by the presence of a //7 chromosome with relative length 8.0% and centromere position 0.72. If the karyotypes at S120 are divided into two classes, one with #7q longer than 5.0% (double exchange) and the other shorter than 4.0% (normal and single exchange), these classes show highly significant differences in the #3 chromosome ( P < 10-5) whereas, for example, the difference in /41 is not significant (P=0.24). Thus, ICR 2A(H)S120 metaphases contained two significantly different classes of//3 chromosomes, the shorter of which was associated with the longer//7. If the double exchange karyotype is classified by r and the single exchange distinguished from normal by the lengths and centromere positions o f / / 4 and ~/6, then a statistical analysis shows that the differences postulated for the four chromosome arms are significant (Table 3). Furthermore, the analysis indicates that //6 is not the same length in the two new karyotypes; thus it is unlikely that the single exchange was a precursor of the double exchange. Whether there has
C h r o m o s o m e s of a Haploid Frog Cell Line Table 3. Analysis of differences in relative lengths of c h r o m o s o m e arms in I C R 2A(H)S120 Chromosome
3q 4p 6q 7q
Karyotype %
Differences
Normal (S.D.)
Single Double exchange exchange (S.D.) (S.D.)
Normal vs. single exchange
Normal vs. double exchange
Single vs. double exchange
8.3(0.6) 4.7(0.6) 4.0(0.6) 3.1 (0.3)
9.1(0.8) 2.6(0.3) 5.2(0.3) 3.4(0.3)
+0.8 -2.1 +1.2 +0.3
--1.6 -1.6 +2.5 +2.7
2.4 0.5 1.3 2.4
6.7(0.5) 3.1(0.6) 6.5(0.7) 5.8(0.5)
P=10 -2 P
