- Email: [email protected]
In vivo BUdR labeling of mammalian chromosomes
396 Preliminary
notes
sensitive DNA fractions correspond to 16. Kornberg, R D, Science 184 (1974) 868. Oosterhof, D K, Hozier, J C & Rill, R L, Proc these two DNA structural compartments, 17. natl acad sci US 72 (1975) 633. respectively. If this is so, the proteins exReceived November 26, 1975 tractable with 0.25 N NaCl are expected to Accepted March 15, 1976 interact both, with nucleosomes as well as with the strands of DNA between the nucleosomes. In the studies to be reported (in preparation) we observe a difference in DNA denaturation between various cell types (i.e. In vivo BUdR labeling of mammalian lymphocytes vs leukemic cells, or between chromosomes various leukemias). These differences, E. L. SCHNEIDER, J. R. CHAILLET and R. R. however, remain after extraction of cells TICE, Laboratory of Cellular and Comparative Phywith 0.25 N NaCl. Thus, the proteins ex- siology, Gerontology Research Center, National Inon Aging, National Institutes of Health, tractable with 0.25 N NaCl do not appear stitute USPHS, US Department of Health, Education and to be the sole, or even the main factor in Welfare, Bethesda and Baltimore City Hospitals, modulating the thermal stability of DNA in Baltimore, MD 21224, USA situ that is responsible for the differences Summary. A technique is described for labeling mammalian chromosomes in vivo with BUdR. Rats and between cell types. mice are given BUdR by tail vein infusion over a 24-h The authors thank Miss Robin Nager for assistance in the preparation of this manuscript. This work was supported by NC1 Grant I-26-CA14134through the National Bladder Cancer Project.
References 1. Lin, J C, Nicolini, C & Baserga, R, Biochemistry 13 (1974) 4127. 2. Nicolini, C, Sally, N G & Baserga, R, Proc natl acad sci US 72 (1975) 2361. 3. Augenlicht. L & Baserea. R. Transnlant nroc 5 (19173)117;. 4. Baserga, R, Life sci 15 (1974) 1057. 5. Kushch, A A, Kolesnikov, V A & Zelenin, A V, Exp cell res 86 (1974) 419. 6. Dariynkiewicz, Z. Traganos, F, Sharpless, T & Melamed, M R, Exp cell res 90 (1975) 411. 7. - J cell bio168 (1976) 1. 8. Melamed, M R & Kamentsky, L A, Int rev exp path01 14 (1974) 205. 9. Bradley, D R & Wolf, M K, Proc natl acad sii us 45 (1959) 944. 10. Rigier, R Jr, Acta physiol Stand, suppl. 67 (1%6) 1. 11. Lerman, L S, Proc natl acad sci US 49 (1963) 94. 12. Dariynkiewicz, Z, Traganos, F, Sharpless, T & Melamed. M R. Pulse-cvtochemistrv (ed C A M Haanen, H F i Hillen & J M C Bessel) p. 88. European Press, Medlcon, Ghent (1975). 13. Nicolini, C & Baserga, R, Arch biochem biophys 169 (1975) 678. 14. Olins. A L & Olins. D E. Science 183(1974) 330. 15. Noll, M, Nature 2jl (1974) 249. I,
Exp Cell Res 100 (1976)
.
_
period at a concentration of 25 pg/g wt/h. Metaphase cells that have gone through two or three cycles of DNA synthesis reveal characteristic differential chromatid fiuorescence after staining with Hoechst dye. Sister chromatid exchanges can then be easily detected in these cells.
The recent advent of the bromodeoxyuridine (BUdR) labeling technique has enabled cytogeneticists to easily detect sisterchromatid exchanges (SCE) in cultured mammalian cells [3, 43. This technique, first developed by Latt [3, 41, involves the substitution of BUdR for thymidine (TdR) in cellular DNA. After two replication cycles in the presence of BUdR, mammalian chromosomes are comprised of one bifilarly BUdR-substituted chromatid and one unifilarly BUdR-substituted chromatid. When metaphase chromosomes are stained with fluorescent dyes, such as Hoechst 33258 or acridine orange, these two types of sister chromatids can be distinguished by their differential fluorescence with the bifilarly. BUdR-substituted chromatid staining less intensely than the unifilarly BUdRsubstituted chromatid (figs 1, 2). SCE can
Preliminary notes 397
Fig. I. Typical rat bone marrow metaphase cells after (a) two and (b) three replication cycles in the presence of BUdR. Fig. 2. Typical mouse bone marrow metaphase cells after (a) two and (6) three replication cycles in the
presence of BUdR. The speckling of the dull-staining chromatids in (a) indicates that the incorporation of BUdR commenced during the period of DNA synthesis.
398 Preliminary
notes
describe the use of the BUdR technique for labeling mammalian chromosomes and detecting SCE in vivo. Materials
and Methods
BUdR in phosphate-buffered saline, pH 7.0, at a concentration of 100-120 ualg wt/h, was administered to 12-month old Wistar random-bred rats and 3-monthold C57B1/6 mice bv tail vein infusion. During the 24 h infusion, mice and rats were restrained in-specialty designed cages seen in fig. 3. In these enclosures, the animals had access to food and water without interfering with the intravenous administration of BUdR. Two hours prior to termination of the BUdR infusion, Colcemid (GIBCo) was injected intravenously at a concentration of 0.1 ml/10 g body weight. Animals infused for as long as 48 h showed no deleterious effects. The animals were sacrificed by cervical dislocation and bone marrow removed from both femurs by washing with PBS. Bone marrow cells were centrifuged at 200 g for 10 mitt; the pelleted cells were suspended in 0.06 M KC1 for either 30 min (mice) or 4.5 mitt (rats) at 37°C. and then recentrifuged at 200 g for 10 min. The pelleted cells were fixed in methanol/ acetic acid (3 : 1) and slides were prepared and stained as previously described [5].
Results and Discussion
Typical rat and mouse metaphase bone marrow cells are seen in figs 1 and 2. Cells after two replication cycles in the presence of BUdR revealed the characteristic differFig. 3. Enclosures designed for containing (a) rats and ential fluorescence seen in vitro with one (b) mice during the period of BUdR infusion by tail bright and one dull chromatid (figs 1a, 2a). vein (modified Bollman restrainers [7]). In metaphase cells after three replication cycles, approximately one-quarter of the total chromatid length fluoresces intensely then be identified by reciprocal exchanges (figs lb, 26). SCE are observed quite of fluorescent intensities (note arrows in easily in these cells (arrows in figs 1, 2). figs 1, 2). The frequencies of SCE in rat and mouse In contrast to previous methods, this bone marrow preparations are listed in table technique has the advantage of being able 1. In addition, table 1 reveals the distributo detect SCE without radioisotope label- tion of cells which have gone through one, ing. This advantage has permitted inves- two or three replication cycles in the prestigators to utilize the BUdR labeling tech- ence of BUdR. It can be seen that in this nique to examine models for chromosome dose range, after a 24 h infusion period, the structure [5]. Although both [3H]TdR and vast majority of metaphase cells have comBUdR have been widely employed to pleted two replication cycles. It has not been completely resolved demonstrate SCE in vitro, the existence of SCE has not been documented in mam- whether SCE are spontaneous or whether malian cells in vivo. In this report, we will they are the result of either the BUdR Exp
Cd
Res
100 (1976)
Preliminary notes 399 Table 1. Frequencies of SCE and the distribution of 1st, 2nd and 3rd replication cycle cells in mouse and rat bone marrow preparations Cell replication cycle” Animal
BUdR dose (/a/g wtlh)
Frequency of SCE” (Mean fS.E.)
Mouse
119
Rat
100 122
1st
2nd
3rd
4.13kO.46
2
89
9
6.83f0.45 8.37k0.49
5 10
i:
,”
(L SCE were scored in thirty 2nd replication cycle cells. b The number of cells in the various cell replication cycles were determined on consecutive metaphase cells.
utilized to detect them or the in vitro culture conditions [2, 61. Of particular importance is the known induction of SCE by BUdR in the presence of light [l]. Since BUdR labeling, as well as addition of mitotic inhibitors, was performed in vivo and SCE were still observed, it is likely that neither in vitro culture conditions nor light are the sole causes of the observed SCE. Further experiments are being pursued to determine the relationship between in vivo BUdR concentration and SCE. Regardless of whether SCE are spontaneous or induced by the BUdR employed to identify them, this in vivo system has several potential applications. It can be utilized to analyse the cell cycle transit times of cell populations in vivo. In addition, it can be used to assess the effects of agents capable of damaging DNA and inducing SCE in vivo.
Cell fusion induced by lysolecithin and concanavalin A in Drosophila melanogaster somatic cells cultured in vitro CARLOTTA
HALFER and LUCIA PETRELLA, In-
stitute of Genetics
of Milan,
Milan, Italy
Cell fusion and homokaryon formation were induced with lysolecithin and concanavahn A (ConA) in two karyotypically different embryonic cell lines of Dr. melanogaster, GM 1 and 185. The fusion capacity and the cytotoxic activity of LL, analysed immediately after treatment, were more rapid and drastic than those of ConA. Moreover, the two lines considered consistently differed in their sensitivity to the chemical agents: GM 1 cells were more sensitive and less efficient in fusion than LB5 cells.
Summary.
During the last 15 years, cell fusion phenomenon has been successfully utilized for some particularly interesting problems in the biological field, such as formal genetics (essentially in man) and cellular function. The best known inducers of cell fusion at present may be classified as follows: (i) biological factors as some inactivated viruses, and in particular the Sendai virus; (ii) We thank Dr Nathaniel Pierce and Mr Maurice E. Zimmerman for technical assistance. chemicals such as lysolecithin (a lysophosphatide) and some phytoagglutinins, i.e. References concanavalin A (ConA) and wheat germ ag1. Ikushima, T &Wolff, S, Exp cell res 87 (1974) 15. 2. Kato, H, Nature 25 1 (1974) 70. glutinin (WGA). All these agents can induce 3. Latt, S A, Proc natl acad sci US 70 (1973) 3395. cell fusion (with homo-heterokaryons and 4. - Science 185 (1974) 74. 5. Tice, R, Chaillet, J & Schneider, E L, Nature 256 hybrid formation) in hen erythrocytes as (1975) 642. well as in several different mammalian cells, 6. Wolff, S & Perry, P, Chromosoma 48 (1974) 341. with the exception of plant lectins which 7. Bollman, J L, J lab clin med 33 (1948) 1348. cause only agglutination between conReceived December 2, 1975 Accepted March 15, 1976 tiguous cells. Exp Cell Res 100 (1976)
notes
sensitive DNA fractions correspond to 16. Kornberg, R D, Science 184 (1974) 868. Oosterhof, D K, Hozier, J C & Rill, R L, Proc these two DNA structural compartments, 17. natl acad sci US 72 (1975) 633. respectively. If this is so, the proteins exReceived November 26, 1975 tractable with 0.25 N NaCl are expected to Accepted March 15, 1976 interact both, with nucleosomes as well as with the strands of DNA between the nucleosomes. In the studies to be reported (in preparation) we observe a difference in DNA denaturation between various cell types (i.e. In vivo BUdR labeling of mammalian lymphocytes vs leukemic cells, or between chromosomes various leukemias). These differences, E. L. SCHNEIDER, J. R. CHAILLET and R. R. however, remain after extraction of cells TICE, Laboratory of Cellular and Comparative Phywith 0.25 N NaCl. Thus, the proteins ex- siology, Gerontology Research Center, National Inon Aging, National Institutes of Health, tractable with 0.25 N NaCl do not appear stitute USPHS, US Department of Health, Education and to be the sole, or even the main factor in Welfare, Bethesda and Baltimore City Hospitals, modulating the thermal stability of DNA in Baltimore, MD 21224, USA situ that is responsible for the differences Summary. A technique is described for labeling mammalian chromosomes in vivo with BUdR. Rats and between cell types. mice are given BUdR by tail vein infusion over a 24-h The authors thank Miss Robin Nager for assistance in the preparation of this manuscript. This work was supported by NC1 Grant I-26-CA14134through the National Bladder Cancer Project.
References 1. Lin, J C, Nicolini, C & Baserga, R, Biochemistry 13 (1974) 4127. 2. Nicolini, C, Sally, N G & Baserga, R, Proc natl acad sci US 72 (1975) 2361. 3. Augenlicht. L & Baserea. R. Transnlant nroc 5 (19173)117;. 4. Baserga, R, Life sci 15 (1974) 1057. 5. Kushch, A A, Kolesnikov, V A & Zelenin, A V, Exp cell res 86 (1974) 419. 6. Dariynkiewicz, Z. Traganos, F, Sharpless, T & Melamed, M R, Exp cell res 90 (1975) 411. 7. - J cell bio168 (1976) 1. 8. Melamed, M R & Kamentsky, L A, Int rev exp path01 14 (1974) 205. 9. Bradley, D R & Wolf, M K, Proc natl acad sii us 45 (1959) 944. 10. Rigier, R Jr, Acta physiol Stand, suppl. 67 (1%6) 1. 11. Lerman, L S, Proc natl acad sci US 49 (1963) 94. 12. Dariynkiewicz, Z, Traganos, F, Sharpless, T & Melamed. M R. Pulse-cvtochemistrv (ed C A M Haanen, H F i Hillen & J M C Bessel) p. 88. European Press, Medlcon, Ghent (1975). 13. Nicolini, C & Baserga, R, Arch biochem biophys 169 (1975) 678. 14. Olins. A L & Olins. D E. Science 183(1974) 330. 15. Noll, M, Nature 2jl (1974) 249. I,
Exp Cell Res 100 (1976)
.
_
period at a concentration of 25 pg/g wt/h. Metaphase cells that have gone through two or three cycles of DNA synthesis reveal characteristic differential chromatid fiuorescence after staining with Hoechst dye. Sister chromatid exchanges can then be easily detected in these cells.
The recent advent of the bromodeoxyuridine (BUdR) labeling technique has enabled cytogeneticists to easily detect sisterchromatid exchanges (SCE) in cultured mammalian cells [3, 43. This technique, first developed by Latt [3, 41, involves the substitution of BUdR for thymidine (TdR) in cellular DNA. After two replication cycles in the presence of BUdR, mammalian chromosomes are comprised of one bifilarly BUdR-substituted chromatid and one unifilarly BUdR-substituted chromatid. When metaphase chromosomes are stained with fluorescent dyes, such as Hoechst 33258 or acridine orange, these two types of sister chromatids can be distinguished by their differential fluorescence with the bifilarly. BUdR-substituted chromatid staining less intensely than the unifilarly BUdRsubstituted chromatid (figs 1, 2). SCE can
Preliminary notes 397
Fig. I. Typical rat bone marrow metaphase cells after (a) two and (b) three replication cycles in the presence of BUdR. Fig. 2. Typical mouse bone marrow metaphase cells after (a) two and (6) three replication cycles in the
presence of BUdR. The speckling of the dull-staining chromatids in (a) indicates that the incorporation of BUdR commenced during the period of DNA synthesis.
398 Preliminary
notes
describe the use of the BUdR technique for labeling mammalian chromosomes and detecting SCE in vivo. Materials
and Methods
BUdR in phosphate-buffered saline, pH 7.0, at a concentration of 100-120 ualg wt/h, was administered to 12-month old Wistar random-bred rats and 3-monthold C57B1/6 mice bv tail vein infusion. During the 24 h infusion, mice and rats were restrained in-specialty designed cages seen in fig. 3. In these enclosures, the animals had access to food and water without interfering with the intravenous administration of BUdR. Two hours prior to termination of the BUdR infusion, Colcemid (GIBCo) was injected intravenously at a concentration of 0.1 ml/10 g body weight. Animals infused for as long as 48 h showed no deleterious effects. The animals were sacrificed by cervical dislocation and bone marrow removed from both femurs by washing with PBS. Bone marrow cells were centrifuged at 200 g for 10 mitt; the pelleted cells were suspended in 0.06 M KC1 for either 30 min (mice) or 4.5 mitt (rats) at 37°C. and then recentrifuged at 200 g for 10 min. The pelleted cells were fixed in methanol/ acetic acid (3 : 1) and slides were prepared and stained as previously described [5].
Results and Discussion
Typical rat and mouse metaphase bone marrow cells are seen in figs 1 and 2. Cells after two replication cycles in the presence of BUdR revealed the characteristic differFig. 3. Enclosures designed for containing (a) rats and ential fluorescence seen in vitro with one (b) mice during the period of BUdR infusion by tail bright and one dull chromatid (figs 1a, 2a). vein (modified Bollman restrainers [7]). In metaphase cells after three replication cycles, approximately one-quarter of the total chromatid length fluoresces intensely then be identified by reciprocal exchanges (figs lb, 26). SCE are observed quite of fluorescent intensities (note arrows in easily in these cells (arrows in figs 1, 2). figs 1, 2). The frequencies of SCE in rat and mouse In contrast to previous methods, this bone marrow preparations are listed in table technique has the advantage of being able 1. In addition, table 1 reveals the distributo detect SCE without radioisotope label- tion of cells which have gone through one, ing. This advantage has permitted inves- two or three replication cycles in the prestigators to utilize the BUdR labeling tech- ence of BUdR. It can be seen that in this nique to examine models for chromosome dose range, after a 24 h infusion period, the structure [5]. Although both [3H]TdR and vast majority of metaphase cells have comBUdR have been widely employed to pleted two replication cycles. It has not been completely resolved demonstrate SCE in vitro, the existence of SCE has not been documented in mam- whether SCE are spontaneous or whether malian cells in vivo. In this report, we will they are the result of either the BUdR Exp
Cd
Res
100 (1976)
Preliminary notes 399 Table 1. Frequencies of SCE and the distribution of 1st, 2nd and 3rd replication cycle cells in mouse and rat bone marrow preparations Cell replication cycle” Animal
BUdR dose (/a/g wtlh)
Frequency of SCE” (Mean fS.E.)
Mouse
119
Rat
100 122
1st
2nd
3rd
4.13kO.46
2
89
9
6.83f0.45 8.37k0.49
5 10
i:
,”
(L SCE were scored in thirty 2nd replication cycle cells. b The number of cells in the various cell replication cycles were determined on consecutive metaphase cells.
utilized to detect them or the in vitro culture conditions [2, 61. Of particular importance is the known induction of SCE by BUdR in the presence of light [l]. Since BUdR labeling, as well as addition of mitotic inhibitors, was performed in vivo and SCE were still observed, it is likely that neither in vitro culture conditions nor light are the sole causes of the observed SCE. Further experiments are being pursued to determine the relationship between in vivo BUdR concentration and SCE. Regardless of whether SCE are spontaneous or induced by the BUdR employed to identify them, this in vivo system has several potential applications. It can be utilized to analyse the cell cycle transit times of cell populations in vivo. In addition, it can be used to assess the effects of agents capable of damaging DNA and inducing SCE in vivo.
Cell fusion induced by lysolecithin and concanavalin A in Drosophila melanogaster somatic cells cultured in vitro CARLOTTA
HALFER and LUCIA PETRELLA, In-
stitute of Genetics
of Milan,
Milan, Italy
Cell fusion and homokaryon formation were induced with lysolecithin and concanavahn A (ConA) in two karyotypically different embryonic cell lines of Dr. melanogaster, GM 1 and 185. The fusion capacity and the cytotoxic activity of LL, analysed immediately after treatment, were more rapid and drastic than those of ConA. Moreover, the two lines considered consistently differed in their sensitivity to the chemical agents: GM 1 cells were more sensitive and less efficient in fusion than LB5 cells.
Summary.
During the last 15 years, cell fusion phenomenon has been successfully utilized for some particularly interesting problems in the biological field, such as formal genetics (essentially in man) and cellular function. The best known inducers of cell fusion at present may be classified as follows: (i) biological factors as some inactivated viruses, and in particular the Sendai virus; (ii) We thank Dr Nathaniel Pierce and Mr Maurice E. Zimmerman for technical assistance. chemicals such as lysolecithin (a lysophosphatide) and some phytoagglutinins, i.e. References concanavalin A (ConA) and wheat germ ag1. Ikushima, T &Wolff, S, Exp cell res 87 (1974) 15. 2. Kato, H, Nature 25 1 (1974) 70. glutinin (WGA). All these agents can induce 3. Latt, S A, Proc natl acad sci US 70 (1973) 3395. cell fusion (with homo-heterokaryons and 4. - Science 185 (1974) 74. 5. Tice, R, Chaillet, J & Schneider, E L, Nature 256 hybrid formation) in hen erythrocytes as (1975) 642. well as in several different mammalian cells, 6. Wolff, S & Perry, P, Chromosoma 48 (1974) 341. with the exception of plant lectins which 7. Bollman, J L, J lab clin med 33 (1948) 1348. cause only agglutination between conReceived December 2, 1975 Accepted March 15, 1976 tiguous cells. Exp Cell Res 100 (1976)