Humangenetik 28, 97--102 (1975) © by Springer-Verlag 1975
Premature Chromosome Condensation in the Bone Marrow of Chinese Hamsters after Whole Body Irradiation with Co6°y ]lays in vivo S. K i i r t e n Hahn-Meitner-Institut fiir Kernforschung Berlin GmbH, Bereich Datenverarbeitung und Elektronik, Berlin G. O b e Institut fiir Genetik, Freie Universit~t Berlin, Berlin Received February 27, 1975
Summary. In the Chinese hamster bone marrow chromosomal aberrations were induced after whole body irradiation with Co 6° y rays in vivo. Aberrant mitoses give rise to lagging chromatin that forms micronnclei. Eventually the micronuclei are slowed down in their cell cycles in comparison te the main nuclei. The mitotic chromatin of the main nuclei induces premature chromosome condensation (PCC) in the micronuclei that are still in the interphase state of their cell cycles. Zusammen/assung. Im Knochenmark des Chinesischen Hamsters wurden chromosomMe Aberrationen nach GanzkSrperbestrahlung mit Co s° y-Strahlen in vivo induziert. Aberrante Mitosen fiihren fiber die Bildung von Chromatinfragmenten zur Entstehung yon Mikrokernen. Die Mikrokerne k6nnen in ihrem Zellcyclus hinter dem der Hauptkerne zurfickbleiben. Das mitotische Chromatin der Hauptkerne induziert in den Mikrokernen, die sich noch in der Interphase befinden, vorzeitige Chromosomenkondensation.
Introduction M i c r o n u c l e i f o r m e d f r o m l a g g i n g c h r o m a t i n m a y be slowed d o w n in t h e i r cell cycle w i t h r e s p e c t to t h e i r a s s o c i a t e d m a i n nuclei, a n d u n d e r t h e i n f l u e n c e o f t h e m i t o s i n g c h r o m a t i n of t h e l a t t e r t h e y m a y u n d e r g o p r e m a t u r e c h r o m o s o m e cond e n s a t i o n (PCC). P C C of t h i s t y p e can be i n d u c e d in vivo a n d in vitro ( I k e u c h i , 1973; K a t o a n d S a n d b e r g , 1968; K i i r t e n a n d Obe, 1975; O b e et al., 1975a, b). I n t h i s c o m m u n i c a t i o n we w a n t t o s h o w t h a t P C C can be i n d u c e d in t h e b o n e m a r r o w of Chinese h a m s t e r s a f t e r w h o l e b o d y i r r a d i a t i o n w i t h Co 6° y r a y s in vivo.
])Iaterials a n d Methods Female Chinese hamsters (2n -- 22), 8 months old and with a body weight of ca. 30.0 g were used. The irradiation was performed with a Co s° y source with an activity of 1000 Ci. The animals were placed in plastic containers of 10.0 cm in length and a diameter of 2.5 cm. The containers were closed with an iron plug on the buttom to increase the stability of the containers. At a distance of I0.0 em the containers with the animals rotated around the Co 6° rod and around themselves in a kind of planetary gearing. This procedure allows whole body
98
S. Kfirten and G. 0be
irradiation to be performed. The irradiation time was 8.5 rain corresponding a dose of 380 rad. The dose was measured with a TOSHIBA dosimeter (Model No. FD-P8; 8.0 × 8.0 × 4.7 ram) located in the plastic containers together with the animals. The dose determination was performed 24 hrs after the irradiation in a fluoro glass dosimeter type FGD-6 (Tokyo Shibura Electric, Ltd.). After irradiation the animals were allowed to recover for 16, 20, 24, 28, and 32 hrs. One animal was used for each recovery time. 4 hrs before the end of the recovery period the animals received an tip. injection of 0.3 mg colehicine in 0.5 ml of a 0.9% NaC1 solution. The animal with 28 hrs recovery time received, 2 ~ hrs before sacrificing, 50 ~xCitritiated thymidine [(3H)TdR] with a specific activity of 52 Ci/mM (Biochemical Center Amersham) for the autoradiographic analysis of DNA synthesis. One animal with a recovery time of 32 hrs was used for the preparation of anaphases and received neither eolchicine nor (3I-I)TdR. Direct chromosome preparations from the bone marrow were performed according to the method of Schmid et al. (1971) and Schmid (1972a, b). The autoradiographies were performed as described (Kfirten and Obe, 1975). The mitoses were analyzed with respect to isoehromatid breaks (excluding those associated with exchanges), ehromatid transloeations, dieentric and ring chromosomes, polyploid mitoses (endoreduplications and tetraploid cells), and PCC. The frequencies of cells with micronuclei and the mitotic indices were also determined. Achromatic lesions and open ehromatid breaks were not scored. In the (3H)TdR series, the PCCs and the nuclei with mieronuelei were analyzed with respect to their labeling pattern.
R e s u l t s a n d Discussion T h e r e s u l t s can be seen in T a b l e 1 a n d in Figs. 1 t o 3. W i t h all r e c o v e r y t i m e s we f o u n d d i e e n t r i e c h r o m o s o m e s a n d r i n g c h r o m o s o m e s (Fig. 2 b a n d g) as well as i s o c h r o m a t i d a b e r r a t i o n s . P o l y e e n t r i e c h r o m o s o m e s w e r e f o u n d in v e r y few cases (Fig. 2 b) ; t h e y w e r e i n c l u d e d in t h e g r o u p o f cells w i t h c h r o m o s o m e t r a n s l o c a t i o n s (see T a b l e 1). U p t o 24 hrs r e c o v e r y t i m e , m i t o s e s w i t h c h r o m a t i d t r a n s l o e a t i o n s (Fig. 2a) a n d in a few cases w i t h c h r o m a t i d a n d c h r o m o s o m e t r a n s l o c a t i o n s can be f o u n d in t h e s a m e cell, i n d i c a t i n g t h a t t h e s e cells h a d b e e n in S - p h a s e a t t h e t i m e o f i r r a d i a t i o n a n d e x h i b i t e d t h e i r first p o s t - i r r a d i a t i o n m i t o s i s (X1). Fig. 1 shows t h e f r e q u e n c i e s of a b e r r a n t m i t o s e s , p o l y p l o i d m i t o s e s , a n d of m i t o s e s w i t h P C C f r o m t h e cells a n a l y z e d in T a b l e 1, t h e f r e q u e n c i e s o f m i c r o n u c l e i c a l c u l a t e d f r o m a n a l y z i n g 1500 i n t e r p h a s e cells in e a c h e n t r y , a n d t h e m i t o t i c indices in p e r c e n t c a l c u l a t e d f r o m t h e a n a l y s i s of t h e f o l l o w i n g n u m b e r s of cells: 16 hrs = 1536; 20 hrs = 1163; 24 hrs - - 1086; 28 hrs = 1143; 32 hrs
Table 1. Exchange-type aberrations and isochromatid breaks (without fragments associated with exchanges) induced in Chinese hamster bone marrow in vivo after whole body irradiation with Co 6° ~, rays in a dose of 380 rad and at recovery times of 16, 20, 24, 28, and 32hrs Recovery time (hrs)
16 20 24 28 32
Number of Chromatid mitoses transanalyzed locations
1115 741 978 917 972
Chromatid and chromosome translocations
Isochromatid breaks
% (total)
Chromosome translocations (dicentric and ring chromosomes) % (total)
% (total)
% (total)
14.26 (159) 4.45 (33) 1.33 (13) ---
18.74 26.18 17.38 18.10 19.75
0.18 (2) 0.27 (2) 0.31 (3) --
19.46 22.94 15.54 12.14 12.55
(209) (194) (170) (166) (192)
(271) (170) (152) (111) (122)
Premature Chromosome Condensation after Irradiation
99
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Premature Chromosome Condensation in the Bone Marrow of Chinese Hamsters after Whole Body Irradiation with Co6°y ]lays in vivo S. K i i r t e n Hahn-Meitner-Institut fiir Kernforschung Berlin GmbH, Bereich Datenverarbeitung und Elektronik, Berlin G. O b e Institut fiir Genetik, Freie Universit~t Berlin, Berlin Received February 27, 1975
Summary. In the Chinese hamster bone marrow chromosomal aberrations were induced after whole body irradiation with Co 6° y rays in vivo. Aberrant mitoses give rise to lagging chromatin that forms micronnclei. Eventually the micronuclei are slowed down in their cell cycles in comparison te the main nuclei. The mitotic chromatin of the main nuclei induces premature chromosome condensation (PCC) in the micronuclei that are still in the interphase state of their cell cycles. Zusammen/assung. Im Knochenmark des Chinesischen Hamsters wurden chromosomMe Aberrationen nach GanzkSrperbestrahlung mit Co s° y-Strahlen in vivo induziert. Aberrante Mitosen fiihren fiber die Bildung von Chromatinfragmenten zur Entstehung yon Mikrokernen. Die Mikrokerne k6nnen in ihrem Zellcyclus hinter dem der Hauptkerne zurfickbleiben. Das mitotische Chromatin der Hauptkerne induziert in den Mikrokernen, die sich noch in der Interphase befinden, vorzeitige Chromosomenkondensation.
Introduction M i c r o n u c l e i f o r m e d f r o m l a g g i n g c h r o m a t i n m a y be slowed d o w n in t h e i r cell cycle w i t h r e s p e c t to t h e i r a s s o c i a t e d m a i n nuclei, a n d u n d e r t h e i n f l u e n c e o f t h e m i t o s i n g c h r o m a t i n of t h e l a t t e r t h e y m a y u n d e r g o p r e m a t u r e c h r o m o s o m e cond e n s a t i o n (PCC). P C C of t h i s t y p e can be i n d u c e d in vivo a n d in vitro ( I k e u c h i , 1973; K a t o a n d S a n d b e r g , 1968; K i i r t e n a n d Obe, 1975; O b e et al., 1975a, b). I n t h i s c o m m u n i c a t i o n we w a n t t o s h o w t h a t P C C can be i n d u c e d in t h e b o n e m a r r o w of Chinese h a m s t e r s a f t e r w h o l e b o d y i r r a d i a t i o n w i t h Co 6° y r a y s in vivo.
])Iaterials a n d Methods Female Chinese hamsters (2n -- 22), 8 months old and with a body weight of ca. 30.0 g were used. The irradiation was performed with a Co s° y source with an activity of 1000 Ci. The animals were placed in plastic containers of 10.0 cm in length and a diameter of 2.5 cm. The containers were closed with an iron plug on the buttom to increase the stability of the containers. At a distance of I0.0 em the containers with the animals rotated around the Co 6° rod and around themselves in a kind of planetary gearing. This procedure allows whole body
98
S. Kfirten and G. 0be
irradiation to be performed. The irradiation time was 8.5 rain corresponding a dose of 380 rad. The dose was measured with a TOSHIBA dosimeter (Model No. FD-P8; 8.0 × 8.0 × 4.7 ram) located in the plastic containers together with the animals. The dose determination was performed 24 hrs after the irradiation in a fluoro glass dosimeter type FGD-6 (Tokyo Shibura Electric, Ltd.). After irradiation the animals were allowed to recover for 16, 20, 24, 28, and 32 hrs. One animal was used for each recovery time. 4 hrs before the end of the recovery period the animals received an tip. injection of 0.3 mg colehicine in 0.5 ml of a 0.9% NaC1 solution. The animal with 28 hrs recovery time received, 2 ~ hrs before sacrificing, 50 ~xCitritiated thymidine [(3H)TdR] with a specific activity of 52 Ci/mM (Biochemical Center Amersham) for the autoradiographic analysis of DNA synthesis. One animal with a recovery time of 32 hrs was used for the preparation of anaphases and received neither eolchicine nor (3I-I)TdR. Direct chromosome preparations from the bone marrow were performed according to the method of Schmid et al. (1971) and Schmid (1972a, b). The autoradiographies were performed as described (Kfirten and Obe, 1975). The mitoses were analyzed with respect to isoehromatid breaks (excluding those associated with exchanges), ehromatid transloeations, dieentric and ring chromosomes, polyploid mitoses (endoreduplications and tetraploid cells), and PCC. The frequencies of cells with micronuclei and the mitotic indices were also determined. Achromatic lesions and open ehromatid breaks were not scored. In the (3H)TdR series, the PCCs and the nuclei with mieronuelei were analyzed with respect to their labeling pattern.
R e s u l t s a n d Discussion T h e r e s u l t s can be seen in T a b l e 1 a n d in Figs. 1 t o 3. W i t h all r e c o v e r y t i m e s we f o u n d d i e e n t r i e c h r o m o s o m e s a n d r i n g c h r o m o s o m e s (Fig. 2 b a n d g) as well as i s o c h r o m a t i d a b e r r a t i o n s . P o l y e e n t r i e c h r o m o s o m e s w e r e f o u n d in v e r y few cases (Fig. 2 b) ; t h e y w e r e i n c l u d e d in t h e g r o u p o f cells w i t h c h r o m o s o m e t r a n s l o c a t i o n s (see T a b l e 1). U p t o 24 hrs r e c o v e r y t i m e , m i t o s e s w i t h c h r o m a t i d t r a n s l o e a t i o n s (Fig. 2a) a n d in a few cases w i t h c h r o m a t i d a n d c h r o m o s o m e t r a n s l o c a t i o n s can be f o u n d in t h e s a m e cell, i n d i c a t i n g t h a t t h e s e cells h a d b e e n in S - p h a s e a t t h e t i m e o f i r r a d i a t i o n a n d e x h i b i t e d t h e i r first p o s t - i r r a d i a t i o n m i t o s i s (X1). Fig. 1 shows t h e f r e q u e n c i e s of a b e r r a n t m i t o s e s , p o l y p l o i d m i t o s e s , a n d of m i t o s e s w i t h P C C f r o m t h e cells a n a l y z e d in T a b l e 1, t h e f r e q u e n c i e s o f m i c r o n u c l e i c a l c u l a t e d f r o m a n a l y z i n g 1500 i n t e r p h a s e cells in e a c h e n t r y , a n d t h e m i t o t i c indices in p e r c e n t c a l c u l a t e d f r o m t h e a n a l y s i s of t h e f o l l o w i n g n u m b e r s of cells: 16 hrs = 1536; 20 hrs = 1163; 24 hrs - - 1086; 28 hrs = 1143; 32 hrs
Table 1. Exchange-type aberrations and isochromatid breaks (without fragments associated with exchanges) induced in Chinese hamster bone marrow in vivo after whole body irradiation with Co 6° ~, rays in a dose of 380 rad and at recovery times of 16, 20, 24, 28, and 32hrs Recovery time (hrs)
16 20 24 28 32
Number of Chromatid mitoses transanalyzed locations
1115 741 978 917 972
Chromatid and chromosome translocations
Isochromatid breaks
% (total)
Chromosome translocations (dicentric and ring chromosomes) % (total)
% (total)
% (total)
14.26 (159) 4.45 (33) 1.33 (13) ---
18.74 26.18 17.38 18.10 19.75
0.18 (2) 0.27 (2) 0.31 (3) --
19.46 22.94 15.54 12.14 12.55
(209) (194) (170) (166) (192)
(271) (170) (152) (111) (122)
Premature Chromosome Condensation after Irradiation
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