Printed in Sweden Copyrighl @ 1977 by Academic Press, Inc. All rights of reproduction in any formreserved ISSN WI44827
PRELIMINARY
NOTES
Selective endoreduplication or branched chromosome? B. NOEL, BERNADETTE QUACK, JOSIANE MOTTET. YVETTE NANTOIS and B. DUTRILLAUX,’ Lhboratorie de CytogtWtique, 73011 Chambkry and ‘Institut de ProgknPse, Paris, France
Among the rare variants of human chromosomes, some have been observed which cause a high frequency of chromatid gaps in particular locations (fig. 1). Occasionally, the gaps are accompanied by triradial figures, described at first under the term selective selective endoreduplication by Lejeune et al. [5]. An alternative explanation for these triradials, based on the malsegregation of the segment distal to the gap has also been proposed [3,4]. The two hypotheses seem mutually exclusive. To test their validity, we have used BUdR to indicate the number of S phases undergone by each chromosome segment after the start of the treatment. Material and Methods The study has been exclusively carried out on the propositus, which shows l-2 triradial figures involving chromosome 2 per 100 mitoses. BUdR has been used for 7-70 h, either from the beginning of culture or added after 48 h in standard culture medium. The final concentration is 10 pg/ml medium. Giemsa staining was used to screen for triradial figures, and acridine orange to demonstrate BUdR incorporation [l]. The total survey comprises almost 10000 mitoses from 33 separate cultures.
Type 1. Segmentation with R banding (fig. 2), indicating BUdR incorporated at the end of the last S phase. Type 2. Chromosome asymmetry, with a short green chromatid and a long red chromatid, indicating BUdR incorporation since the beginning of the previous S phase. Type 3. Partial asymmetry, with statistically half of the chromosomes as type 2 and the other half red and long for both chromatids, indicating BUdR incorporation for three S phases. Among the type 1 cells, we have seen one triradial figure (fig. 3). Among type 2 cells, eight triradial figures were found and among type 3 cells three only. In type 2 cells, the two long arms of the abnormal no. 2 chromosome are similar, one chromatid of each being green and the other red. This is the same as the appearance of the other chromosomes. In type 3 cells, the staining of long arms of no. 2 chromosome varies. One has one green and one red chromatid, the other has two red chromatids. As for the short arm of no. 2, both chromatids are red or one green and the other red. To summarize, the appearance of the
Results
The chromatid gaps have been seen every time. The frequency of triradial figures seems much lower after BUdR treatment than in normal medium. To try to simplify our results as much as possible, we describe three types of A0 staining: 28-761813
Fig. 1. Chromosome no. 2 with gap q deletion, radial and bead-like figures. Exp
Cell
Res
104
tri-
(1977)
424
Preliminary
notes
Fig. 2. A0 staining. Type 1, segmentation with R banding; type 2, chromosome asymmetry; type 3, partial asymmetry.
abnormal no. 2 chromosome is equivalent to that of the other chromosomes. A more detailed analysis of these figures will be reported elsewhere. Discussion
The staining with acridine orange is not only related to the amount of BUdR incorporation in chromosome DNA [2]. However, it may be accepted that every green fluorescing chromatid has at least one DNA strand without BUdR. When the BUdR treatment is given during
Fig.
3. Triradial figures, type 1, 2, 3.
Exp Cell Res 104 (1977)
the last two S phases, every chromosome will have one green chromatid (with one normal and one substituted strand) and one red chromatid (with two substituted DNA strands). When the treatment is given during three consecutive S phases, half the chromosomes will be as described above, and the other half, which has double substituted DNA strands, will be completely red. It is possible to check the hypothesis of selective endoreduplication by study of triradials in cells which have incorporated
Preliminary
notes
425
We can now check if the hypothesis of malsegregation of the long arm of no. 2 chromosome agrees with the observation. The different segregations of an independent long arm against the centric fragment are considered in fig. 4. After three S phases, two types of triradial figures are theoretically possible. Out of three mitoses showing triradial figures, we have seen these two possible figures. This result agrees well with the tested hypothesis. Furthermore, in one of the triradials, the green chromatid of the long arm is distributed between two chromosome fragments, indicating a sister chromatid exchange at the penultimate generation. Therefore, there is truly one cell transmission of two sister chromatid segments, which indicates malsegregation. Fig. 4. Schematic drawings of triradials in cells which have incorporated BUdR during S phases. (a) After supposed endoreduplication; (b) after break and nondisjunction.
BUdR during S phases. If the long arm of no. 2 had been endoreduplicated, it would have been exposed to BUdR during one S phase more, and it would have one green chromatid and three red ones (fig. 4). In fact, we have never observed this appearance. The triradial figures have as many green as red chromatids. When the long arms of the triradial figure present one green for three red chromatids, the whole mitosis shows statistically one quarter of green for three quarters of red. It may be concluded that the long arms of the triradial figure have, in every case, undergone the same number of S phases as the whole mitosis. The hypothesis of selective endoreduplication is not compatible with this result, except under one particular set of circumstances that the endoreduplicaions occurred in vivo, before addition of 3UdR.
Conclusion
The triradials occasionally observed in a patient with a 2 qh+ do not result from selective endoreduplication of the no. 2 long arm, but of mitotic malsegregation. The malsegregation is probably the consequence of a fragile no. 2 chromosome and this interesting structural peculiarity is being studied. It is worth mentioning that the site of observed qh+ seem to recall ancient Simian chromosome rearrangements:telomeric fusion of two acrocentrics. We are indebted to Mrs Petit, the generous 2 qh patient, and to Dr Bobrow for suggestions and help. This work has been supported by RCP 85 CNRS.
References 1. Dutrillaux, B, Laurent, C, Couturier, J & Lejeune, J, Compt rend acad sci 276 (1973) 3179. 2. Dutrillaux, B, Aurias, A & Fosse, A M, Exp cell res 97 (1976) 313. 3. Ferguson-Smith, M A, Ann g&ret 16 (1973) 29. 4. Ford, C E & Madan, K, Chromosome identificacion-technique and appfications (ed T Caspersson & L Zech). Proc 23 Nobel Symposium, StockExp Cell Res 104 (1977)
426
Preliminary notes
holm (1972), p. 98. Nobel Foundation, Stockholm, and Academic Press, New York (1973). 5. Lejeune, J, Dutrillaux, B, Lafourcade, J, Berger, R, Abonyi, D & Rethore, M D, Compt rend acad sci 266 (1968) 24. Received May 3 1, 1976 Accepted June 10, 1976
Proliferation of serum-deprived neuroblastoma cells K. W. LANKS, Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA Summary. Both the mitotic index after colchicine block and the fraction of cells labeled by a short exposure to [3H]TdR indicate that the rate of proliferation in cultures of murine neuroblastoma cells (N18 clone) decreases only after several days of maintenance in serumdeftcient medium. Viable cell counts remain constant because ot a large increase in the number of cells which die and are shed into the medium. Therefore, the neurite formation and enzyme induction which occur under these conditions cannot be caused by inhibition of DNA synthesis.
they increase their rate of AChE synthesis [8], we noticed that although the number of viable cells per culture indeed remained fairly constant, dead cells appeared continuously in the supernatant culture medium. Further examination of this phenomenon revealed that since proliferation of cultured neuroblastoma cells is largely independent of serum factors the neurite extension that occurs under conditions of serum deprivation cannot cause inhibition of proliferation. On the other hand, maintenance of viability does appear to depend on serum concentration and the implications of this situation are discussed. Materials and Methods Neuroblastoma cells were maintained in 10% or O.l %-0.2% fetal bovine serum as previously described [8]. Monolayer cells were removed in saline-D, [l] and cells in the medium were concentrated by brief centrifugation. Preliminary experiments showed that in the range of O.l-OS% serum there was little or no increase in the number of viable cells per culture while the absolute amount and specific activity of AChE in the monolayer cells increased as previously reported [8]. Viability of cell suspensions was determined by Trypan Blue exclusion and counting in a hemocytometer. Pelleted cells were also smeared onto glass slides, air-dried, fixed in methanol/acetic acid (3 : 1) and Giemsa stained. Viability of monolayer cells was determined after comparison with similarly prepared supematant cells from serum-deprived cmtures, over 99% of which failed to exclude Trypan Blue. Mitotic indices were obtained from smears made after exposure of cultures to 0.5 fig/ml colchicine for 2 h. Only cells with clearly condensed chromosomes and dissolution of the nuclear membrane were considered to be mitotic. Cells were labeled with [SH]TdR (61 Cilmmole) for 30 min after varying periods of culture in serum-deficient medium, smears were made after thorough washing with medium and autoradiography performed [9] using Kodak NTB2 emulsion.
The observation that when cultured cells are maintained in serum-deficient medium they cease to proliferate and acquire more mature morphological and biochemical characteristics is reported in the earliest studies of the murine neuroblastoma system [l-3]. Subsequently, inhibition of proliferation by various means [4, 51 has been shown to enhance neurite extension and acetylcholinesterase (AChE) synthesis. Despite evidence against the inverse relationship [3,6], i.e., neurite extension necessarily precludes proliferation, it has been suggested that effecters of such morphological changes might be used therapeuti- Results cally [7]. The initial impression that proliferation The association of neurite extension with continued in serum-deprived cultures was apparent inhibition of proliferation under confirmed in the experiment described in conditions of serum deprivation may have table 1 where the number of cells/culture allowed the impression of a causal relation- increased from 1.5X 1oJto 23X 1oJover the ship to persist. While showing that when 5 day course. Most of this increase was due neuroblastoma cells are deprived of serum to the accumulation of dead cells. The dead Exp CeNRes 104 (1977)
PRELIMINARY
NOTES
Selective endoreduplication or branched chromosome? B. NOEL, BERNADETTE QUACK, JOSIANE MOTTET. YVETTE NANTOIS and B. DUTRILLAUX,’ Lhboratorie de CytogtWtique, 73011 Chambkry and ‘Institut de ProgknPse, Paris, France
Among the rare variants of human chromosomes, some have been observed which cause a high frequency of chromatid gaps in particular locations (fig. 1). Occasionally, the gaps are accompanied by triradial figures, described at first under the term selective selective endoreduplication by Lejeune et al. [5]. An alternative explanation for these triradials, based on the malsegregation of the segment distal to the gap has also been proposed [3,4]. The two hypotheses seem mutually exclusive. To test their validity, we have used BUdR to indicate the number of S phases undergone by each chromosome segment after the start of the treatment. Material and Methods The study has been exclusively carried out on the propositus, which shows l-2 triradial figures involving chromosome 2 per 100 mitoses. BUdR has been used for 7-70 h, either from the beginning of culture or added after 48 h in standard culture medium. The final concentration is 10 pg/ml medium. Giemsa staining was used to screen for triradial figures, and acridine orange to demonstrate BUdR incorporation [l]. The total survey comprises almost 10000 mitoses from 33 separate cultures.
Type 1. Segmentation with R banding (fig. 2), indicating BUdR incorporated at the end of the last S phase. Type 2. Chromosome asymmetry, with a short green chromatid and a long red chromatid, indicating BUdR incorporation since the beginning of the previous S phase. Type 3. Partial asymmetry, with statistically half of the chromosomes as type 2 and the other half red and long for both chromatids, indicating BUdR incorporation for three S phases. Among the type 1 cells, we have seen one triradial figure (fig. 3). Among type 2 cells, eight triradial figures were found and among type 3 cells three only. In type 2 cells, the two long arms of the abnormal no. 2 chromosome are similar, one chromatid of each being green and the other red. This is the same as the appearance of the other chromosomes. In type 3 cells, the staining of long arms of no. 2 chromosome varies. One has one green and one red chromatid, the other has two red chromatids. As for the short arm of no. 2, both chromatids are red or one green and the other red. To summarize, the appearance of the
Results
The chromatid gaps have been seen every time. The frequency of triradial figures seems much lower after BUdR treatment than in normal medium. To try to simplify our results as much as possible, we describe three types of A0 staining: 28-761813
Fig. 1. Chromosome no. 2 with gap q deletion, radial and bead-like figures. Exp
Cell
Res
104
tri-
(1977)
424
Preliminary
notes
Fig. 2. A0 staining. Type 1, segmentation with R banding; type 2, chromosome asymmetry; type 3, partial asymmetry.
abnormal no. 2 chromosome is equivalent to that of the other chromosomes. A more detailed analysis of these figures will be reported elsewhere. Discussion
The staining with acridine orange is not only related to the amount of BUdR incorporation in chromosome DNA [2]. However, it may be accepted that every green fluorescing chromatid has at least one DNA strand without BUdR. When the BUdR treatment is given during
Fig.
3. Triradial figures, type 1, 2, 3.
Exp Cell Res 104 (1977)
the last two S phases, every chromosome will have one green chromatid (with one normal and one substituted strand) and one red chromatid (with two substituted DNA strands). When the treatment is given during three consecutive S phases, half the chromosomes will be as described above, and the other half, which has double substituted DNA strands, will be completely red. It is possible to check the hypothesis of selective endoreduplication by study of triradials in cells which have incorporated
Preliminary
notes
425
We can now check if the hypothesis of malsegregation of the long arm of no. 2 chromosome agrees with the observation. The different segregations of an independent long arm against the centric fragment are considered in fig. 4. After three S phases, two types of triradial figures are theoretically possible. Out of three mitoses showing triradial figures, we have seen these two possible figures. This result agrees well with the tested hypothesis. Furthermore, in one of the triradials, the green chromatid of the long arm is distributed between two chromosome fragments, indicating a sister chromatid exchange at the penultimate generation. Therefore, there is truly one cell transmission of two sister chromatid segments, which indicates malsegregation. Fig. 4. Schematic drawings of triradials in cells which have incorporated BUdR during S phases. (a) After supposed endoreduplication; (b) after break and nondisjunction.
BUdR during S phases. If the long arm of no. 2 had been endoreduplicated, it would have been exposed to BUdR during one S phase more, and it would have one green chromatid and three red ones (fig. 4). In fact, we have never observed this appearance. The triradial figures have as many green as red chromatids. When the long arms of the triradial figure present one green for three red chromatids, the whole mitosis shows statistically one quarter of green for three quarters of red. It may be concluded that the long arms of the triradial figure have, in every case, undergone the same number of S phases as the whole mitosis. The hypothesis of selective endoreduplication is not compatible with this result, except under one particular set of circumstances that the endoreduplicaions occurred in vivo, before addition of 3UdR.
Conclusion
The triradials occasionally observed in a patient with a 2 qh+ do not result from selective endoreduplication of the no. 2 long arm, but of mitotic malsegregation. The malsegregation is probably the consequence of a fragile no. 2 chromosome and this interesting structural peculiarity is being studied. It is worth mentioning that the site of observed qh+ seem to recall ancient Simian chromosome rearrangements:telomeric fusion of two acrocentrics. We are indebted to Mrs Petit, the generous 2 qh patient, and to Dr Bobrow for suggestions and help. This work has been supported by RCP 85 CNRS.
References 1. Dutrillaux, B, Laurent, C, Couturier, J & Lejeune, J, Compt rend acad sci 276 (1973) 3179. 2. Dutrillaux, B, Aurias, A & Fosse, A M, Exp cell res 97 (1976) 313. 3. Ferguson-Smith, M A, Ann g&ret 16 (1973) 29. 4. Ford, C E & Madan, K, Chromosome identificacion-technique and appfications (ed T Caspersson & L Zech). Proc 23 Nobel Symposium, StockExp Cell Res 104 (1977)
426
Preliminary notes
holm (1972), p. 98. Nobel Foundation, Stockholm, and Academic Press, New York (1973). 5. Lejeune, J, Dutrillaux, B, Lafourcade, J, Berger, R, Abonyi, D & Rethore, M D, Compt rend acad sci 266 (1968) 24. Received May 3 1, 1976 Accepted June 10, 1976
Proliferation of serum-deprived neuroblastoma cells K. W. LANKS, Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA Summary. Both the mitotic index after colchicine block and the fraction of cells labeled by a short exposure to [3H]TdR indicate that the rate of proliferation in cultures of murine neuroblastoma cells (N18 clone) decreases only after several days of maintenance in serumdeftcient medium. Viable cell counts remain constant because ot a large increase in the number of cells which die and are shed into the medium. Therefore, the neurite formation and enzyme induction which occur under these conditions cannot be caused by inhibition of DNA synthesis.
they increase their rate of AChE synthesis [8], we noticed that although the number of viable cells per culture indeed remained fairly constant, dead cells appeared continuously in the supernatant culture medium. Further examination of this phenomenon revealed that since proliferation of cultured neuroblastoma cells is largely independent of serum factors the neurite extension that occurs under conditions of serum deprivation cannot cause inhibition of proliferation. On the other hand, maintenance of viability does appear to depend on serum concentration and the implications of this situation are discussed. Materials and Methods Neuroblastoma cells were maintained in 10% or O.l %-0.2% fetal bovine serum as previously described [8]. Monolayer cells were removed in saline-D, [l] and cells in the medium were concentrated by brief centrifugation. Preliminary experiments showed that in the range of O.l-OS% serum there was little or no increase in the number of viable cells per culture while the absolute amount and specific activity of AChE in the monolayer cells increased as previously reported [8]. Viability of cell suspensions was determined by Trypan Blue exclusion and counting in a hemocytometer. Pelleted cells were also smeared onto glass slides, air-dried, fixed in methanol/acetic acid (3 : 1) and Giemsa stained. Viability of monolayer cells was determined after comparison with similarly prepared supematant cells from serum-deprived cmtures, over 99% of which failed to exclude Trypan Blue. Mitotic indices were obtained from smears made after exposure of cultures to 0.5 fig/ml colchicine for 2 h. Only cells with clearly condensed chromosomes and dissolution of the nuclear membrane were considered to be mitotic. Cells were labeled with [SH]TdR (61 Cilmmole) for 30 min after varying periods of culture in serum-deficient medium, smears were made after thorough washing with medium and autoradiography performed [9] using Kodak NTB2 emulsion.
The observation that when cultured cells are maintained in serum-deficient medium they cease to proliferate and acquire more mature morphological and biochemical characteristics is reported in the earliest studies of the murine neuroblastoma system [l-3]. Subsequently, inhibition of proliferation by various means [4, 51 has been shown to enhance neurite extension and acetylcholinesterase (AChE) synthesis. Despite evidence against the inverse relationship [3,6], i.e., neurite extension necessarily precludes proliferation, it has been suggested that effecters of such morphological changes might be used therapeuti- Results cally [7]. The initial impression that proliferation The association of neurite extension with continued in serum-deprived cultures was apparent inhibition of proliferation under confirmed in the experiment described in conditions of serum deprivation may have table 1 where the number of cells/culture allowed the impression of a causal relation- increased from 1.5X 1oJto 23X 1oJover the ship to persist. While showing that when 5 day course. Most of this increase was due neuroblastoma cells are deprived of serum to the accumulation of dead cells. The dead Exp CeNRes 104 (1977)
