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Radiation-induced aneusomic clones in bone marrow of rats
121
Mutation Research, 35 (1976) 121--128 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
R A D I A T I O N J N D U C E D ANEUSOMIC CLONES IN BONE M A R R O W O F RATS
SEI-ICHI KOHNO * and TAKAAKI ISHIHARA
Division of Radiation Health, National Institute of Radiological Sciences, Chiba, 280 (Japan) (Received July 29th, 1975) (Revision received November 14th, 1975) (Accepted November 28th, 1975)
Summary Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats,54 clones of cellswith radiation-induced chromosome abnormalities, ranging from 3.3 to 78.3% in size, were obtained. Karyotype analysis at the banding levelshowed that 43 out of the 54 clones had balanced chromosome constitutions, and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general beliefthat cellswith unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all,except 1, of the largest 6 clones were unbalanced, eitheraneuploid or pseudo
Introduction Chromosome abnormalities in bone marrow of radiation-exposed subjects have been observed in the form of clones o f cells with the same abnormalities [1,7,17--19]. Such a clone originates from a single cell with radiation-induced c h r o m o s o m e abnormalities. In some instances, all the marrow cells are replaced by m e m b e r cells o f a single clone. Although the biological significance o f the * Present addzeu: Depaztment of Biology, Faculty of Science, T o h o Urdvendty, Miyama-cho, Funabsahi, Chiba, 274. Japan.
122 clone-forming cells with radiation-induced chromosome abnormalities is not yet fully known, these cells are believed to have genetically essential chromosome material similar to that of the normal diploid set, with the karyotypes balanced, and therefore to be genetically neutral [4]. This assumption, however, was drawn from observations carried out by the conventional method which has limitations in the positive identification of minor chromosome abnormalities, translocations and inversions. We report here the results of our chromosome analysis of done-forming cells in irradiated rat bone marrow, carried out with recently developed banding techniques that permit positive identification of each chromosome [3]. Our special concern has been to determine whether the chromosome constitution of a clone cell is really balanced, and whether karyotypic features of clone cells have anything to do with their establishment or development as clones, or with the clone size. It was found that, whereas many of the clones studied appeared to have balanced chromosome constitutions, those that were larger were often aneusomic clones with gross chromosome deficiency or excess: a result in contrast with the general expectation of selection against chromosomally unbalanced cells. Materials and methods The animals used were 23 rats 3 months old. The rats were of non-inbred Wistar strain maintained in a closed colony since 1960, when their ancestral inbred Wistar/Ms rats, raised at the National Institute of Genetics, Mishima, Japan, were brought to our Institute. The animals were given a single whole-body X-ray dose of 700 R at a dose rate of 20 R/min. The radiation source was an X-ray machine operated at 200 kVp, 20 m A , filtered by 0.5 m m Cu and 0.5 m m Al, H V L of 1.15 m m Cu. The distance from focus to skin was 100 cm. The rats were killed 5 to 15 months (average, 9.3 months) after irradiation. T w o hours before the rats were killed, 0.1% colchicine, 1 mg/kg body weight, was injected into peritoneal cavities.Bone marrow samples were obtained from femurs, and suspended in 10 ml Hanks' solution with 100 units of heparin. The cells were kept in a 0.06 M KCI hypotonic solution for 10 rain at 37°C, and fixed with acetic alcohol (1 : 3) for 15 min. The preparation of cells was made by a routine air drying method. The banding patterns of the chromosomes of the bone marrow cells were obtained by the trypsin Giemsa technique of Seabright [22] with some modifications, and by the quinacrine fluorescence technique of Mori and Sasaki [15]. The frequency of a clone was indicated by the number of clone cells in all the cells (about 60) analyzed per rat.
Results The number of clones of cells with chromosome abnormalities obtained from the 23 irradiated rats was 54. The size of each clone varied from 3.3 to 78.3%. These 54 different clones were subjected to further karyotypic analysis by banding techniques.
123 TABLE I KARYOTYPES AND FREQUENCIES OF LARGE CLONES FROM BONE MARROW OF X-IRRADIATED RATS Clone
Frequency of c l o n e
Time between irradiation and
Type of clone a
Karyotype of clone
D E D D B D B B D E B B B D B
41, 43, 41, 42, 42, 40, 42, 42, 41, 42, 42, 42, 42, 42, 42,
killing (months) 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15
78.3(47160) 71.7(43/60) 66.7(40]60) 65.0(39160) 58.3(35160) 45.0(27/60) 36.7(22/60) 36.7(22/60) 33.3(20/60) 31.7(19/60) 31.7(19/60) 30.0(18/60)
26.7(16/60) 25.0(15160) 25.0(15/60)
9 13 II 13 7 7 13 12 7 15 7 13 12 7 9
X, Xq--, --17, t ( l q - - ; 9p+) XY, +10, t(8q+; 10q--) XY, --19 XX, t(4q--; 15p+), 9q-XX, t ( l p + ; 5q--), t(Sp+; 18q--) XY, --15, --16, 10p+ XX, t(3q--; 4q+) XY, t ( l q - - ; 2q+) XY, --19, 10p+ XY, 19q+ XY, t(3q--; 15q+) XX, t(6q--; 20q+) XY, t ( l q - - ; 8q+) XX, t(Tq+; 177--), 187-XY, t(7p+; 11q--)
a B, D and E indicate a balanced, a d e l e t e d a n d a n e x c e u i v e t y p e o f c l o n e , respecltvely.
The majority, 43 of the 54 clones (79.6%), showed balanced karyotypes, b u t the remaining 11 clones (20.4%) were of unbalanced karyotypes. The 43 balanced clones consisted of 33 types with reciprocal translocations, 6 types with inversions and 4 types with both reciprocal translocations and inversions. The 11 unbalanced clones were made up of 7 types of aneuploid clones with either loss or addition of one or more chromosomes, and of 4 types of pseudo-diploid clones with aneusomic complement. Table I lists, in order of size, 15 large clones having frequencies of more than 25%. Five of the largest six clones, namely Nos. 1, 2, 3, 4, and 6 were unbalanced. The largest was a clone with 41 chromosomes from which chromosome No. 17 was missing. As Fig. 1 shows, there was a translocation between the long arm of No. 1 and the short arm of No. 9, and there was also a piece missing from the long arm of the X chromosome. The second largest clone, No. 2, had 43 chromosomes. The a d d i t i o n ~ c h r o m o s o m e was identified as No. 10, b u t was a b o u t ~ of the normal length. A b o u t ~ of this chromosome was translocated to the distal end of No. 8 c h r o m o s o m e (Fig. 2). Clone No. 3 had 41 chromosomes, No. 19 being missing. Clone No. 4 was a pseudo
124
Fig. 1. F l u o r e s c e n c e k a z y o t y p e o f t h e l a r g e s t c l o n e ( c l o n e N o . 1) w i t h 4 1 c h r o m o s o m e s . N o , 1 7 c h r o m o s o m e is m i s s i n g ; p a r t o f t h e X c h r o m o s o m e is m i s s i n g ; a n d t h e r e is a t r a n s l o c a t i o n b e t w e e n N o s . 1 a n d 9 c h r o m o s o m e s . 4 1 , X , X q - - , - - 1 7 , t ( l q - - ; 9 p + ).
,..., 1
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8
19i gg
I0
11
12
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F i g . 2: G i e m s a - b a n d i n g k a r y o t y p e o f c l o n e N o . 2 s h o w i n g 4 8 c h r o m o s o m e s w i t h a n a d d i t i o n a l N o . 1 0 chromosome and a translocation between Nos. 8 and 10 chromosomes. 43,XY,+10,t(8q+;10q--).
125
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6
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Fig. 3. G i e m s a - b a n d l n g k a r y o t y p e o f c l o n e N o . 6 s h o w i n g 4 0 c h r o m o s o m e s . C h r o m o s o m e s N o s . 1 5 a n d 1 6 a r e m i s s i n g ; a n d t h e r e is a n a d d i t i o n a l p a r t i n N o . 1 0 . 4 0 , X Y , - - 1 5 , - - 1 6 , 1 0 p + .
the short arm of No. 5 and the long arm of No. 18. Eight of the 15 large clones were of either aneuploid or pseudo
OF THE
F r e q u e n c i e s (%) Balanced Unbalanced Total
54 BALANCED
100--25.0 7
loss addition
6 (4) a 2 (1) a 15
AND UNBALANCED
CLONES IN FOUR
FREQUENCY
24.9--10.0
9.9--5.0
4.9--0
Total
11
14
11
43 (79.6%)
1 (1) a 0 12
a Value in parentheses shows the number of aneuploid clones.
1 0 15
1 (1) a 0 12
9 (16.7%) 2 (3.7%) 54 (100.0%)
126 Discussion More than half of the 15 large clones t h a t occurred with frequencies of over 25% were aneusomic. This finding appears to be inconsistent with the general idea that aneusomic cells are selectively eliminated in favor of cells with balanced karyotypes. Ford [4] has stated that, in irradiated mouse somatic tissues, selection operates in favor of a cell with an essentially complete genotype as opposed to that with a grossly deficient one. In an experiment on isolating aneusomic clones from a Chinese hamster cell line by the induction of non-disjunction, cells with unbalanced monosomic or trisomic chromosome constitution had less capacity to proliferate than had the cells of a parental line [8]. In man, accumulated data on patients with atJnormalities of sex or autosomal chromosomes indicate that a zygote from a germ cell with chromosome abnormalities would n o t be expected to achieve normal development, and t h a t loss of any autosome may lead to death [6]. A high percentage of chromosome abnormalities in spontaneous abortions is well known [5]. Whereas all these data appear to support the expectation of selection against cells with unbalanced chromosome constitutions, especially those with monosomy, there is no evidence for this selection in the present study. Formation of clones with chromosome abnormalities in rat and mouse bone marrow after irradiation is well known [1,17--19]. According to observations by K o h n o et al. [10], in rat bone marrow cells at I day, 1 m o n t h and 4 months after X-irradiation with a dose of 100, 300, 500 and 700 R, clone formation was not observed 1 day after irradiation, when cells with various types of chromosome abnormality existed independently; but at 1 m o n t h , the number of cell types decreased and clone formation was recognized. The tendency became more evident at 4 months when the number of cell types showed a further decrease to only a few, some showing increase in size. The process of clone formation has been more clearly shown in the serial observations in an irradiated rat using bone marrow biopsy [9], in which process a small number of clones is formed, with the rise and fall of each clone, from a variety of cells with radiation-induced chromosome abnormalities, has been revealed. Such formation of clones of cells with chromosome abnormalities should proceed through the recovery process of stem cells in radiation-damaged bone marrow [18]. Radiation-induced aneusomic clones in blood cells have been observed in humans [2,7] and mice [20]. Regarding the formation of aneusomic cells, Russell and Saylors [21] have reported t h a t non-disjunction in sex chromosomes may be induced by radiation in mouse germ cells, and t h a t the types and the frequencies of the abnormalities depend on the cell stages irradiated. On the other hand, there is some evidence that the induction of aneusomic clones is n o t necessarily due to a direct effect of irradiation. In serial m o n t h l y observations in the same rat bone marrow, Kohno et al. [11,12] noticed two cases in which a sudden increase in clone size was recognized 3--4 m o n t h s after irradiation. In both instances, the sudden increase in size was accompanied by a loss of one chromosome from a clone with marked chromosome abnormalities, which was recognized as a new clone with m o n o s o m y in the samples thereafter.
127 TABLE III CHROMOSOMES INVOLVED ANEUSOMIC CLONES
IN
T H E MONOSOMY OR T R I S O M Y ( P A R T I A L OR W H O L E ) OF
C h r o m o s o m e No.
1
9
10
15
16
17
18
19
X
?
Total
Loss
1
1
--
1
1
2
2
2
1
--
11
Addition
--
--
1
.
1
2
.
.
.
.
.
Such change in the chromosome number may be due to non
Acknowledgements We are grateful to Dr. T. Kumatori, National Institute of Radiological Sciences, Chiba, for his interest in this work and for helpful discussion. This work was supported by the project grant for Carcinogenesis Study from the Science and Technology Agency, Japan. References I B a r n e s , D.W.H., C.E. F o r d , S.M. G r a y a n d J . F . L o u t i t , S p o n t a n e o u s and induced changes i n cell populations i n h e a v i l y i r r a d i a t e d m i c e , P r o g r . Nuol. E n e r g y , Set. VI., 2 ( 1 9 5 9 ) 1 - - 1 0 . 2 B e n d e r , M.A. a n d P.C. G o o c h , C h r o m o s o m e aberrations in i r r a d i a t e d h u m a n s , P r o c e e d i n g s X l t h I n t e r n a t i o n a l C o n g r e s s a n R a d i o l o g y , 1 9 6 5 , pp. 1 4 2 1 - - 1 4 2 5 . 3 C o m m i t t e e for a S t a n d a r d i z e d K a r Y o t y p e o f Ruttus norvegicus, S t a n d a r d K a r y o t y p e of the N o r w a y R a t , Ruttus norvegicus, C y t o g e n e t . Cell G e n e t . , 1 2 ( 1 9 7 3 ) 1 9 9 - - 2 0 5 . 4 F o r d , C.E., Selection pressure in m a m m a l i a n cell populations, in R.J.C. H a r r i s (cd.), Cytogenetics of Cells in C u l t u r e , A c a d e m i c Press, N e w York, 1 9 6 4 , p p . 2 7 - - 4 5 .
128 5 Geneva Conference, Standardization of Procedures for C h r o m o s o m e Studies in Abortion, Cytogenetics, 5 (1966) 3 61--393. 6 Hamerton, J.L., H u m a n Cytogenetics, Clinical Cytogenetics, Vol. II, Academic Press, New York, 1971. 7 Ishihara, T. an d T. Kumatori, Cytogenetic studies on fishermen exposed t o fallout ra di a t i on in 1954, Japan. J. Genetics, 44 (1969) 242--251. 8 Kato, H. and T.H. Yosida, Isolation of aneusomic clones from Chinese h a m s t e r cell line following induction of nondisjunctton, Cytogenetics, 10 (1971) 392--403. 9 Kohno, S , S. Koide, T. Ishihara and T. Kumatori, Pursuit of clones of cells w i t h radiation-induced c h r o m o s o m e aberrations in hone marrow of the rat by re pe a t e d ma rrow biopsies, C hromos ome Inform a t i o n Service, 12 (1971) 25--27. 10 Kohno, S , M. Inaba, T. Ishihara and T. Kumatori, Clones of cells w i t h c h r o m o s o m e aberrations in h e m a t o p o i e t i c tissues after irradiation. I. Effect of dose t o the establishment of clones in bone marr o w of rats, Nat. Inst. Radiol. Sci. Ann. Rept., 11 (1972) 33--39. 11 Kohno, S., T. Iehihara and T. K u m a t o r i , Clones of cells with c h r o m o s o m e aberrations in h e m a t o p o i e t ic tissues after irradiation. II. Serial observations in bone ma rrow of X-irradiated rats, Nat. Inst. Radiol. Sci. Ann. Rept., 11 (1972) 39--41. 12 Kohno, S. and T. Ishihara, 1]npublished results. 13 Kurita, Y., T. Sugiyama and Y . / q i s h i z u k a , Cytogenetic studies on rat l e u k e m i a i nduc e d by pulse dose of 7,12-dimethylbenz(a)anthracene, Cancer Res., 28 (1968) 1 7 3 3 - - 1 7 5 2 . 14 Moloney, W.C., A.E. Boschetti and G. Dowd, Observations on l e u k e m i a in Wistar and Wistar Furth rats, Blood, 26 (1965) 341---353. 15 Moil, M. and M. Sasaki, Fluorescence banding patterns of the rat c hromos ome s , Chromosoma, 40 (1973) 173--182. 16 Mori, M. and M. Sasaki, C h r o m o s o m e studies o n rat leukemias and ] y m p h o m a s , with special attention to fluorescent ks.ryotype analysis, J. ~ it. Cancer Inst., 52 (1974) 153--160. 17 Howell, P.C., R e d u c e d incidence of persistent c h r o m o s o m e aberrations in mice irradiated at l ow dose rates, Science 141 (1963) 524--526. 18 Howell, P.C. an d L.J. Cole, Clonal r e p o p n i a t i o n in reticular tissues of X-irradiated mice: Effect of dose and of limb-shielding, J. Cell. Physiol., 70 (1967) 37---44. 19 Nowel], P.C., B.E. Hirsch, D.H. Fox and D.B. Wilson, Evidence for t he existence of m u l t i p o t e n t i a l l y m p h o - h e m a t o p o i e t i e stem cells in the a d u l t rat, J. Cell. Physiol., 75 (1970) 151--158. 20 Howell, P.C., D.A. Hungerford and L.J. Cole, C h r o m o s o m e changes following irradiation in mammals, Ann. N.Y. Acad. Sci., 114 (1964) 252--257. 21 Russell, L.B. and C.L. Saylors, The relative sensitivity of various germ-cell stages of t he mouse t o radiation-induced non-disjunction, c h r o m o s o m e losses and deficiencies, in F.H. Sobels (ed.), Repair from Genetic Radiation Damage, Pergamon Press, London, 1963, pp. 313--340. 22 Seabright, M., A rapid banding t e c h n i q u e for h u m a n chromos ome s , Lancet, ii (1971) 971--972.
Mutation Research, 35 (1976) 121--128 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
R A D I A T I O N J N D U C E D ANEUSOMIC CLONES IN BONE M A R R O W O F RATS
SEI-ICHI KOHNO * and TAKAAKI ISHIHARA
Division of Radiation Health, National Institute of Radiological Sciences, Chiba, 280 (Japan) (Received July 29th, 1975) (Revision received November 14th, 1975) (Accepted November 28th, 1975)
Summary Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats,54 clones of cellswith radiation-induced chromosome abnormalities, ranging from 3.3 to 78.3% in size, were obtained. Karyotype analysis at the banding levelshowed that 43 out of the 54 clones had balanced chromosome constitutions, and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general beliefthat cellswith unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all,except 1, of the largest 6 clones were unbalanced, eitheraneuploid or pseudo
Introduction Chromosome abnormalities in bone marrow of radiation-exposed subjects have been observed in the form of clones o f cells with the same abnormalities [1,7,17--19]. Such a clone originates from a single cell with radiation-induced c h r o m o s o m e abnormalities. In some instances, all the marrow cells are replaced by m e m b e r cells o f a single clone. Although the biological significance o f the * Present addzeu: Depaztment of Biology, Faculty of Science, T o h o Urdvendty, Miyama-cho, Funabsahi, Chiba, 274. Japan.
122 clone-forming cells with radiation-induced chromosome abnormalities is not yet fully known, these cells are believed to have genetically essential chromosome material similar to that of the normal diploid set, with the karyotypes balanced, and therefore to be genetically neutral [4]. This assumption, however, was drawn from observations carried out by the conventional method which has limitations in the positive identification of minor chromosome abnormalities, translocations and inversions. We report here the results of our chromosome analysis of done-forming cells in irradiated rat bone marrow, carried out with recently developed banding techniques that permit positive identification of each chromosome [3]. Our special concern has been to determine whether the chromosome constitution of a clone cell is really balanced, and whether karyotypic features of clone cells have anything to do with their establishment or development as clones, or with the clone size. It was found that, whereas many of the clones studied appeared to have balanced chromosome constitutions, those that were larger were often aneusomic clones with gross chromosome deficiency or excess: a result in contrast with the general expectation of selection against chromosomally unbalanced cells. Materials and methods The animals used were 23 rats 3 months old. The rats were of non-inbred Wistar strain maintained in a closed colony since 1960, when their ancestral inbred Wistar/Ms rats, raised at the National Institute of Genetics, Mishima, Japan, were brought to our Institute. The animals were given a single whole-body X-ray dose of 700 R at a dose rate of 20 R/min. The radiation source was an X-ray machine operated at 200 kVp, 20 m A , filtered by 0.5 m m Cu and 0.5 m m Al, H V L of 1.15 m m Cu. The distance from focus to skin was 100 cm. The rats were killed 5 to 15 months (average, 9.3 months) after irradiation. T w o hours before the rats were killed, 0.1% colchicine, 1 mg/kg body weight, was injected into peritoneal cavities.Bone marrow samples were obtained from femurs, and suspended in 10 ml Hanks' solution with 100 units of heparin. The cells were kept in a 0.06 M KCI hypotonic solution for 10 rain at 37°C, and fixed with acetic alcohol (1 : 3) for 15 min. The preparation of cells was made by a routine air drying method. The banding patterns of the chromosomes of the bone marrow cells were obtained by the trypsin Giemsa technique of Seabright [22] with some modifications, and by the quinacrine fluorescence technique of Mori and Sasaki [15]. The frequency of a clone was indicated by the number of clone cells in all the cells (about 60) analyzed per rat.
Results The number of clones of cells with chromosome abnormalities obtained from the 23 irradiated rats was 54. The size of each clone varied from 3.3 to 78.3%. These 54 different clones were subjected to further karyotypic analysis by banding techniques.
123 TABLE I KARYOTYPES AND FREQUENCIES OF LARGE CLONES FROM BONE MARROW OF X-IRRADIATED RATS Clone
Frequency of c l o n e
Time between irradiation and
Type of clone a
Karyotype of clone
D E D D B D B B D E B B B D B
41, 43, 41, 42, 42, 40, 42, 42, 41, 42, 42, 42, 42, 42, 42,
killing (months) 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15
78.3(47160) 71.7(43/60) 66.7(40]60) 65.0(39160) 58.3(35160) 45.0(27/60) 36.7(22/60) 36.7(22/60) 33.3(20/60) 31.7(19/60) 31.7(19/60) 30.0(18/60)
26.7(16/60) 25.0(15160) 25.0(15/60)
9 13 II 13 7 7 13 12 7 15 7 13 12 7 9
X, Xq--, --17, t ( l q - - ; 9p+) XY, +10, t(8q+; 10q--) XY, --19 XX, t(4q--; 15p+), 9q-XX, t ( l p + ; 5q--), t(Sp+; 18q--) XY, --15, --16, 10p+ XX, t(3q--; 4q+) XY, t ( l q - - ; 2q+) XY, --19, 10p+ XY, 19q+ XY, t(3q--; 15q+) XX, t(6q--; 20q+) XY, t ( l q - - ; 8q+) XX, t(Tq+; 177--), 187-XY, t(7p+; 11q--)
a B, D and E indicate a balanced, a d e l e t e d a n d a n e x c e u i v e t y p e o f c l o n e , respecltvely.
The majority, 43 of the 54 clones (79.6%), showed balanced karyotypes, b u t the remaining 11 clones (20.4%) were of unbalanced karyotypes. The 43 balanced clones consisted of 33 types with reciprocal translocations, 6 types with inversions and 4 types with both reciprocal translocations and inversions. The 11 unbalanced clones were made up of 7 types of aneuploid clones with either loss or addition of one or more chromosomes, and of 4 types of pseudo-diploid clones with aneusomic complement. Table I lists, in order of size, 15 large clones having frequencies of more than 25%. Five of the largest six clones, namely Nos. 1, 2, 3, 4, and 6 were unbalanced. The largest was a clone with 41 chromosomes from which chromosome No. 17 was missing. As Fig. 1 shows, there was a translocation between the long arm of No. 1 and the short arm of No. 9, and there was also a piece missing from the long arm of the X chromosome. The second largest clone, No. 2, had 43 chromosomes. The a d d i t i o n ~ c h r o m o s o m e was identified as No. 10, b u t was a b o u t ~ of the normal length. A b o u t ~ of this chromosome was translocated to the distal end of No. 8 c h r o m o s o m e (Fig. 2). Clone No. 3 had 41 chromosomes, No. 19 being missing. Clone No. 4 was a pseudo
124
Fig. 1. F l u o r e s c e n c e k a z y o t y p e o f t h e l a r g e s t c l o n e ( c l o n e N o . 1) w i t h 4 1 c h r o m o s o m e s . N o , 1 7 c h r o m o s o m e is m i s s i n g ; p a r t o f t h e X c h r o m o s o m e is m i s s i n g ; a n d t h e r e is a t r a n s l o c a t i o n b e t w e e n N o s . 1 a n d 9 c h r o m o s o m e s . 4 1 , X , X q - - , - - 1 7 , t ( l q - - ; 9 p + ).
,..., 1
2//
3
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I0
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F i g . 2: G i e m s a - b a n d i n g k a r y o t y p e o f c l o n e N o . 2 s h o w i n g 4 8 c h r o m o s o m e s w i t h a n a d d i t i o n a l N o . 1 0 chromosome and a translocation between Nos. 8 and 10 chromosomes. 43,XY,+10,t(8q+;10q--).
125
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2
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8
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10
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6
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Fig. 3. G i e m s a - b a n d l n g k a r y o t y p e o f c l o n e N o . 6 s h o w i n g 4 0 c h r o m o s o m e s . C h r o m o s o m e s N o s . 1 5 a n d 1 6 a r e m i s s i n g ; a n d t h e r e is a n a d d i t i o n a l p a r t i n N o . 1 0 . 4 0 , X Y , - - 1 5 , - - 1 6 , 1 0 p + .
the short arm of No. 5 and the long arm of No. 18. Eight of the 15 large clones were of either aneuploid or pseudo
OF THE
F r e q u e n c i e s (%) Balanced Unbalanced Total
54 BALANCED
100--25.0 7
loss addition
6 (4) a 2 (1) a 15
AND UNBALANCED
CLONES IN FOUR
FREQUENCY
24.9--10.0
9.9--5.0
4.9--0
Total
11
14
11
43 (79.6%)
1 (1) a 0 12
a Value in parentheses shows the number of aneuploid clones.
1 0 15
1 (1) a 0 12
9 (16.7%) 2 (3.7%) 54 (100.0%)
126 Discussion More than half of the 15 large clones t h a t occurred with frequencies of over 25% were aneusomic. This finding appears to be inconsistent with the general idea that aneusomic cells are selectively eliminated in favor of cells with balanced karyotypes. Ford [4] has stated that, in irradiated mouse somatic tissues, selection operates in favor of a cell with an essentially complete genotype as opposed to that with a grossly deficient one. In an experiment on isolating aneusomic clones from a Chinese hamster cell line by the induction of non-disjunction, cells with unbalanced monosomic or trisomic chromosome constitution had less capacity to proliferate than had the cells of a parental line [8]. In man, accumulated data on patients with atJnormalities of sex or autosomal chromosomes indicate that a zygote from a germ cell with chromosome abnormalities would n o t be expected to achieve normal development, and t h a t loss of any autosome may lead to death [6]. A high percentage of chromosome abnormalities in spontaneous abortions is well known [5]. Whereas all these data appear to support the expectation of selection against cells with unbalanced chromosome constitutions, especially those with monosomy, there is no evidence for this selection in the present study. Formation of clones with chromosome abnormalities in rat and mouse bone marrow after irradiation is well known [1,17--19]. According to observations by K o h n o et al. [10], in rat bone marrow cells at I day, 1 m o n t h and 4 months after X-irradiation with a dose of 100, 300, 500 and 700 R, clone formation was not observed 1 day after irradiation, when cells with various types of chromosome abnormality existed independently; but at 1 m o n t h , the number of cell types decreased and clone formation was recognized. The tendency became more evident at 4 months when the number of cell types showed a further decrease to only a few, some showing increase in size. The process of clone formation has been more clearly shown in the serial observations in an irradiated rat using bone marrow biopsy [9], in which process a small number of clones is formed, with the rise and fall of each clone, from a variety of cells with radiation-induced chromosome abnormalities, has been revealed. Such formation of clones of cells with chromosome abnormalities should proceed through the recovery process of stem cells in radiation-damaged bone marrow [18]. Radiation-induced aneusomic clones in blood cells have been observed in humans [2,7] and mice [20]. Regarding the formation of aneusomic cells, Russell and Saylors [21] have reported t h a t non-disjunction in sex chromosomes may be induced by radiation in mouse germ cells, and t h a t the types and the frequencies of the abnormalities depend on the cell stages irradiated. On the other hand, there is some evidence that the induction of aneusomic clones is n o t necessarily due to a direct effect of irradiation. In serial m o n t h l y observations in the same rat bone marrow, Kohno et al. [11,12] noticed two cases in which a sudden increase in clone size was recognized 3--4 m o n t h s after irradiation. In both instances, the sudden increase in size was accompanied by a loss of one chromosome from a clone with marked chromosome abnormalities, which was recognized as a new clone with m o n o s o m y in the samples thereafter.
127 TABLE III CHROMOSOMES INVOLVED ANEUSOMIC CLONES
IN
T H E MONOSOMY OR T R I S O M Y ( P A R T I A L OR W H O L E ) OF
C h r o m o s o m e No.
1
9
10
15
16
17
18
19
X
?
Total
Loss
1
1
--
1
1
2
2
2
1
--
11
Addition
--
--
1
.
1
2
.
.
.
.
.
Such change in the chromosome number may be due to non
Acknowledgements We are grateful to Dr. T. Kumatori, National Institute of Radiological Sciences, Chiba, for his interest in this work and for helpful discussion. This work was supported by the project grant for Carcinogenesis Study from the Science and Technology Agency, Japan. References I B a r n e s , D.W.H., C.E. F o r d , S.M. G r a y a n d J . F . L o u t i t , S p o n t a n e o u s and induced changes i n cell populations i n h e a v i l y i r r a d i a t e d m i c e , P r o g r . Nuol. E n e r g y , Set. VI., 2 ( 1 9 5 9 ) 1 - - 1 0 . 2 B e n d e r , M.A. a n d P.C. G o o c h , C h r o m o s o m e aberrations in i r r a d i a t e d h u m a n s , P r o c e e d i n g s X l t h I n t e r n a t i o n a l C o n g r e s s a n R a d i o l o g y , 1 9 6 5 , pp. 1 4 2 1 - - 1 4 2 5 . 3 C o m m i t t e e for a S t a n d a r d i z e d K a r Y o t y p e o f Ruttus norvegicus, S t a n d a r d K a r y o t y p e of the N o r w a y R a t , Ruttus norvegicus, C y t o g e n e t . Cell G e n e t . , 1 2 ( 1 9 7 3 ) 1 9 9 - - 2 0 5 . 4 F o r d , C.E., Selection pressure in m a m m a l i a n cell populations, in R.J.C. H a r r i s (cd.), Cytogenetics of Cells in C u l t u r e , A c a d e m i c Press, N e w York, 1 9 6 4 , p p . 2 7 - - 4 5 .
128 5 Geneva Conference, Standardization of Procedures for C h r o m o s o m e Studies in Abortion, Cytogenetics, 5 (1966) 3 61--393. 6 Hamerton, J.L., H u m a n Cytogenetics, Clinical Cytogenetics, Vol. II, Academic Press, New York, 1971. 7 Ishihara, T. an d T. Kumatori, Cytogenetic studies on fishermen exposed t o fallout ra di a t i on in 1954, Japan. J. Genetics, 44 (1969) 242--251. 8 Kato, H. and T.H. Yosida, Isolation of aneusomic clones from Chinese h a m s t e r cell line following induction of nondisjunctton, Cytogenetics, 10 (1971) 392--403. 9 Kohno, S , S. Koide, T. Ishihara and T. Kumatori, Pursuit of clones of cells w i t h radiation-induced c h r o m o s o m e aberrations in hone marrow of the rat by re pe a t e d ma rrow biopsies, C hromos ome Inform a t i o n Service, 12 (1971) 25--27. 10 Kohno, S , M. Inaba, T. Ishihara and T. Kumatori, Clones of cells w i t h c h r o m o s o m e aberrations in h e m a t o p o i e t i c tissues after irradiation. I. Effect of dose t o the establishment of clones in bone marr o w of rats, Nat. Inst. Radiol. Sci. Ann. Rept., 11 (1972) 33--39. 11 Kohno, S., T. Iehihara and T. K u m a t o r i , Clones of cells with c h r o m o s o m e aberrations in h e m a t o p o i e t ic tissues after irradiation. II. Serial observations in bone ma rrow of X-irradiated rats, Nat. Inst. Radiol. Sci. Ann. Rept., 11 (1972) 39--41. 12 Kohno, S. and T. Ishihara, 1]npublished results. 13 Kurita, Y., T. Sugiyama and Y . / q i s h i z u k a , Cytogenetic studies on rat l e u k e m i a i nduc e d by pulse dose of 7,12-dimethylbenz(a)anthracene, Cancer Res., 28 (1968) 1 7 3 3 - - 1 7 5 2 . 14 Moloney, W.C., A.E. Boschetti and G. Dowd, Observations on l e u k e m i a in Wistar and Wistar Furth rats, Blood, 26 (1965) 341---353. 15 Moil, M. and M. Sasaki, Fluorescence banding patterns of the rat c hromos ome s , Chromosoma, 40 (1973) 173--182. 16 Mori, M. and M. Sasaki, C h r o m o s o m e studies o n rat leukemias and ] y m p h o m a s , with special attention to fluorescent ks.ryotype analysis, J. ~ it. Cancer Inst., 52 (1974) 153--160. 17 Howell, P.C., R e d u c e d incidence of persistent c h r o m o s o m e aberrations in mice irradiated at l ow dose rates, Science 141 (1963) 524--526. 18 Howell, P.C. an d L.J. Cole, Clonal r e p o p n i a t i o n in reticular tissues of X-irradiated mice: Effect of dose and of limb-shielding, J. Cell. Physiol., 70 (1967) 37---44. 19 Nowel], P.C., B.E. Hirsch, D.H. Fox and D.B. Wilson, Evidence for t he existence of m u l t i p o t e n t i a l l y m p h o - h e m a t o p o i e t i e stem cells in the a d u l t rat, J. Cell. Physiol., 75 (1970) 151--158. 20 Howell, P.C., D.A. Hungerford and L.J. Cole, C h r o m o s o m e changes following irradiation in mammals, Ann. N.Y. Acad. Sci., 114 (1964) 252--257. 21 Russell, L.B. and C.L. Saylors, The relative sensitivity of various germ-cell stages of t he mouse t o radiation-induced non-disjunction, c h r o m o s o m e losses and deficiencies, in F.H. Sobels (ed.), Repair from Genetic Radiation Damage, Pergamon Press, London, 1963, pp. 313--340. 22 Seabright, M., A rapid banding t e c h n i q u e for h u m a n chromos ome s , Lancet, ii (1971) 971--972.