Biochimica et Biophysica Acta, 1132(1992) 43-48
43
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 92404
Different patterns of gene expression of top0isomerase II isoforms in differentiated tissues during murine development Giovanni Capranico, Stella Tinelli, Caroline A. Austin 1, Mark L. Fisher ' and Franco Zunino Dirision of Experimental Oncolo,~ B, lstituto Nazionale per lo Studio e la Cura dei Tumori, Milan (Italy)
(Received 16 March 1992)
Key words: Topoisomerase isoform; Gene expression; Normal tissue; Differentiation; Development; (Mouse) The expression of DNA topoisomerase II a and /3 genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II a gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II /3 gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the /3 gene expression was observed in the brain but not in the liver; conversely, the expression of the a gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II a may be a specific marker of cell proliferation, whereas topoisomerase II /3 may be implicated in functions of DNA metabolism other than replication.
Introduction D N A topoisomerases II are essential enzymes in living cells playing a role in vital processes, such as D N A replication, transcription, recombination and repair [1,2]. In addition, eukaryotic topoisomerase II is a structural c o m p o n e n t of mitotic c h r o m o s o m e scaffolds [3-5] and the nuclear matrix of interphase cells [6] and has been implicated in sister chromatid exchange [1,2] and in the organisation of D N A loop domains [7]. The nuclear topoisomerase II is a major target of clinically effective antineoplastic drugs [8-10]. These agents reversibly stabilize a normal intermediate formed during the enzyme-catalyzed reaction, resulting in D N A cleavage stimulation. T h e drug action then initiates a complex and poorly understood cascade of events culminat-
Correspondence to: G. Capranico, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. i Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, London, UK.
ing in cell death [1,2,8,9]. Although many factors play a role in the drug cytotoxic activity, one important determinant is the cellular content of topoisomerase II [11-18]. The a m o u n t and activity of topoisomerase II were shown to correlate with the state of cell proliferation in normal cells [19-22], missing in nonproliferative tissues and quiescent cells. The regulation of topoisomerase II levels in t u m o r cells seems to be more complex. Topoisomerase II levels have been reported to be higher in transformed than in normal cells, and not to decline to low levels when cancer cells cease proliferation [23-25]. Thus, topoisomerase II regulation may be altered in transformed as o p p o s e d to normal cells, representing a basis for the anti-tumor activity of topoisomerase II inhibitors. However, topoisomerase II levels in highly proliferating h u m a n hematopoietic progenitor cells were shown to be indistinguishable from levels observed in h u m a n leukemia cell lines [26] and markedly decreased both in m a t u r e hematopoietic cells and in leukemic cells induced to differentiate [26]. O n the other hand, appreciable amounts of topoisomerase II
44 have been described in non-proliferating Drosophila normal tissues [27-29], chicken late spermatids [30], some normal human tissues [31] and in rat neuronal nuclei [32]. Therefore, these observations documented that topoisomerase lI may be present in some terminally differentiated cells, although detailed information is missing on the regulation of topoisomerase II in normal tissues during differentiation. In recent years, human and murine tumor cells have been shown to contain two isoenzymes of topoisomerase II, termed p170 and p180, which have been reported to be pharmacologically and biochemically distinct [33,34]. The two isoforms might be differentially regulated during changes in the proliferation state [35], in ras-trasformed murine cells [23] and in drug-resistant tumor cells [33]. Non-overlapping sets of topoisomerase lI c D N A fragments were recently cloned from human cells, and shown to derive from two distinct genes, a and /3, which likely encode for the two isoforms p170 and p180 of topoisomerase II, respectively [36-38]. Many of the previous investigations on topoisomerase II content in mammalian cells have been performed with western blotting techniques often using antibodies specific for the p170 isoform. The availability of specific D N A probes for the two topoisomerase I1 genes, then, prompted us to investigate the expression of these g e n e s in normal murine tissues in the attempt to understand the functions of D N A topoisomerase II isoforms in normal differentiated cells. The expression of the two genes was examined in thirty normal tissues of adult and newborn mice, and in murine embryos.
Materials and M e t h o d s
cDNA probes As murine topoisomerase II isoform gene probes were not available, human topoisomerase II e D N A fragments were used in this study. Two different e D N A fragments were used to detect specifically the topoisomerase II ~ gene (coding for the p170 form): the h-Top2-Z2 probe [38], corresponding to a 3' terminal portion of the gene, provided by Dr. L.F. Liu; and the p G T O P 2 - 1 probe, corresponding to a 5' terminal portion of the gene, recently cloned from the human NCI-H187 cell line using a PCR-based procedure [17]. The c D N A clone CAA5, obtained from a H e L a cell c D N A library [37], was used to specifically determine the expression of the topoisomerase II /3 gene (coding for the p180 form). As a control probe for RNA loading, a mouse LLRep3 0.8 kb Pst I D N A fragment was derived from plasmid pLLRep3 [39]. The mouse thymidylate synthase probe, a 1 kb Pst I fragment, was derived from plasmid pMTS-3 [40]. RNA extraction and Northern blot analysis C3H mice were purchased from Charles River (Calco, Italy). Mice were maintained and treated following the Principles and Guidelines for the Use of Animals in Research of the Istituto Nazionale per lo Studio e la Cura dei Tumori. Tissues were excised from 3-month-old healthy mice (adult animals) or 1-day-old mice (newborns) and cleaned of surrounding material. Embryos were excised 16, 8 or 2 days before delivery and cleaned of parental tissues. Total R N A was immediately extracted by the LiCl-guanidine monothio-
#.,o
6.2
K b -,,.
6.2
K b .-,-
"4" . . . .
""
_
.o
,r
.........
•
Topo
II 0
T O p O II I~'
Fig. 1. Northern blot analysis of topoisomerase II a and /3 gene expression in tissues of adult mice. Total RNAs were fractionated on a denaturing 1% agarose gel, transferred to nylon membranes and hybridised with the h-Top2-Z2 (upper panels) or CAA5 (lower panels) probes in 50% formamide, 5 x SSC, 0.2% SDS, 5 x Denhardt's solution, 50 mM sodium phosphate (pH 7), 250/zg/ml of salmon sperm DNA, 10% dextran sulphate at 42°C for 20 h. Final washes of filters were at 55°C in 0.I XSSC, which did not result in a cross-hybridization between the a and /3 probes.
45
cyanate method [41]. RNA (20/zg) was fractionated on a formaldehyde-containing 1% agarose gel and then transferred to a nylon membrane (Hybond). The membrane was irradiated with ultraviolet light and prehybridization was for at least 4 h at 42°C in 50% formamide, 5 x SSC, 0.2% sodium dodecyl sulphate (SDS), 5 × Denhardt's solution, 50 mM sodium phosphate (pH 7), 250 tzg/ml of salmon sperm DNA. DNA probes were labelled at a specific activity of 2 to 5 - 10 ~ cpm/Izg DNA with the Multiprime DNA labelling systems and deoxycytidine-5'[a- 32P]triphosphate (3000 Ci mmol -~) (Amersham International, UK). Hybridization was carried out for 20 h at 42°C in the same buffer containing 10% dextran sulphate. Final wash of filters was at 55°C in 0.1 x SSC. Autoradiography was at -70°C for 1 to 7 days.
A
e^ 1
2
n 3
4
a 5
6
e
n
7
8
6.2 Kb.~i
To
. p
6.2
Topo II a
Results
Expression of topoisomerase H isoform genes in adult mice Total RNA was purified from each of 22 normal tissues of 3-month-old mice and examined for topoisomerase II a and /3 gene expression by Northern blots using the h-Top2-Z2 [38] and CAA5 probes [37] (Figs. 1 and 2). These probes detected m R N A species of 6.2 kb in all the tissues studied. The patterns of expression differed markedly among examined tissues, showing that the cDNA probes used specifically recognized distinct transcripts in murine RNA. The a gene was highly expressed in the thymus (Fig. 2A, lane 5; B, lane 8), spleen and marrow (Figs. 1 and 2), and to a lesser extent in the intestine, stomach and testis (Fig. 1). Northern blotting analysis using a second topoisomerase II a probe, pGTOP2-1 [17], gave the same results (not shown). The /3 gene was expressed more evenly and in a wider range of the studied tissues of adult mice (Figs. 1 and 2). In particular, a relatively high expression was observed not only in the spleen and marrow but also in the uterus, ovary, lymph nodes, adrenal gland, eye, bladder and heart (Fig. 1). The expression level of the two genes in each mouse tissue was determined relative to the expression level in the thymus (Table I), this being the tissue in adult mice expressing both genes at the highest levels (Fig. 2). RNA loading on gels was checked by ethidium bromide staining and densitometry of gel photographs. Re-hybridizations of filters with a control DNA probe, LLRep3, was also performed in some cases (not shown). Tissues were ranked in four groups with undetectable, low, intermediate and high expression levels (Table I). The patterns of gene expression were markedly different for the two isoenzymes, with the a gene being expressed in fewer tissues than the /3 gene (5 and 15 tissues, respectively, at 'intermediate' and 'high' levels, Table I). Expression of the a gene in the 'low' group
B
n
a
1 2345678
6.2 Kb'~
Topo II 0
Fig. 2. Topoisomerase II a and /3 gene expression in tissues from newborn mice, embryos and selected tissues of adults. See legend to Fig. 1 for methods. Letters e, n and a indicate embryos, newborns and adults, respectively. (A) Lanes 1-3, total embryos 16, 8 and 2 days before delivery, respectively; 4, lung of newborns; 5, thymus of adults; 6, spleen of adults; 7, heart of embryos 2 days before delivery; 8, heart of newborns. (B) Lanes 1, lung; 2, thymus; 3, heart; 4, breast; 5, ovary; 6, marrow; 7, testis; 8, thymus.
(Table I) was detected on autoradiograms by using longer exposure times than those shown in Figs. 1-3. Thus, topoisomerase II a was expressed mainly in proliferative tissues, while topoisomerase II /3 was also expressed at intermediate levels in many non-proliferating tissues. Of tissues examined in adult mice, only the pancreas, salivary glands and muscle did not show detectable expression of either gene (Table I).
Topoisomerase H gene expression in murine embryos and newborns The a and /3 gene expressions were also examined during murine embryogenesis and in newborns (1-dayold mice), and compared to the thymus and other selected tissues of adult mice. Expression of the/3 gene in whole embryos at 16 days before delivery was higher (2.5-fold) than in the thymus of adult mice, and dropped to low levels in embryos 8 and 2 days before delivery (Fig. 2A, lanes 1-3 and 5). High levels of the a gene expression were also seen in early embryos which dropped in older embryos (Fig. 2A); however, it was
46 TABLE 1
BRAIN
Expression o f topoisomerase 11 a and fl genes in dif]~'rentiated murine tissues
A
r
1
LIVER "I
2
3
r
4
A
5
6
Expression level " undetectable
low
Topoisomerase ll c~ gene kidney, liver, breast, ~ muscle, brain, stomach, lung, pancreas, ()vary, uterus, bladder, heart, lymph nodes, eyes, tongue, saliwlry glands, adrenal glands. Topoisomerase 11 fl genc pancreas, liver, salivary glands, testis, muscle, kidney, brain,
intermediate
high
0.6 intestine, (I.4 testis, 0.4 0.3
2.2 thymus, 1.5 marrow, spleen,
10 4.7 3.9
0.3 0.3 0.2 0.1
2.9 thymus, 2.6 2.5 2.3 2.0 1.9 1.6 1.4 1.3 1.1 1.1 0.9 0.9 0.9
10
adrenal glands. ovary, spleen, lymph nodes, uterus, heart, eyes, marrow, bladder, tongue, lung, stomach, intestine, breast, ~'
" Levels of topoisomerase II gene expression are shown relative to those of thymus, set equal to 10. The data shown were calculated from 2 to 3 independent experiments. h From female mice I day after delivery.
never higher than that in the thymus of adult mice, the tissue with the highest level of a gene expression. The decrease in expression in older embryos could be partly due to a dilution effect, as both genes were expressed in tissues of embryos 2 days before delivery at detectable levels (Fig. 2A, lane 7; Fig. 3, lanes 1 and 4). The thymus, lung, liver, brain and heart were evaluated in 1-day-old mice, and expression of both genes was observed in all these tissues (Figs. 2 and 3). Again, the expression levels of the a and /3 genes were different in the newborn tissues (Table If). The thymus and liver expressed the highest levels of a topoisomerase lI RNA and the brain the lowest one, while levels of /3 gene expression were highest in the brain and lowest in the liver.
Post-natal changes of topoisomerase H gene expression Topoisomerase isoform expression was further compared in the liver and brain during murine development. The a gene was expressed in these tissues from embryos (2 days before delivery) and newborns, but not from adult mice (Fig. 3). An increase in gene expression for this isoform was observed in the newborn
6.2 Kb .-~
Topo II a
6.2 Kb -~
Topo In 13
1.3 K b ~
TS t
Fig. 3. Analysis of topoisomerase 11 ce and fl gene expression in murine brain and liver during development. See legend of Fig. 1 for methods. The filters were further rehybridised with a mouse D N A probe for the thymidylate synthase gene (TS). Lanes 1 and 4, embryos 2 days before delivery: 2 and 5, newborns: 3 and 6, adults.
tissues as compared to the embryo tissues, with a prominent increase in the liver, about 2 to 3-fold (Fig. 3, upper panel). The liver and brain of adult mice expressed low levels of topoisomerase II /3 (Table I); strikingly, a marked increase (more than 6-fold) of expression of the /3 gene was observed in the brain of T A B L E 11 Expression of topoisomerase 11 a and fi genes in tissues ~f newborn mice " Tissue
o~ gene
13 gene
Thymus (adults) Thymus Liver Heart Lung Brain
10 4.1 3.6 1.9 1.6 1.5
10 8.8 2.6 8.2 9.8 18.1
~' l-day-old mice. Levels of topoisomerase II gene expression are shown relative to those of adult mouse thymus, set equal to 10. The data shown were calculated from 2 to 3 independent experiments.
47 newborns as compared to the embryos, but only a slight increase was observed in the case of the liver (Fig. 3, middle panel). These increases in the postnatal period were not correlated with an increase in the cell division rate as evaluated by the expression level of the thymidylate synthase gene (Fig. 3, lower panel), a marker of cell proliferation [42]. For the /3 gene, a similar increase in expression was also observed in the heart of newborn mice as compared to that of embryos (Fig. 2A, lanes 7 and 8). Discussion The present results indicate that topoisomerase II and /3 genes are differentially expressed in normal murine tissues. Among 22 tissues from adult (3-monthold) mice, the a gene expression was essentially restricted to five tissues, while the/3 gene was expressed in 15 tissues at high or intermediate levels (Table I). The thymus was the tissue that expressed both genes at the highest level in adult mice. Moreover, in several tissues only the /3 gene expression was detectable (heart, adrenal glands, lymph nodes, uterus, eyes, ovary, bladder, tongue and lung). The c~ gene was expressed in tissues characterized by a relatively high fraction of replicating cells, suggesting a main role of the p170 isoenzyme in functions associated with D N A replication. In contrast, the /3 gene was also expressed in tissues not expected to have proliferating cells, suggesting that the /3 isoform may also participate in D N A metabolic processes other than D N A synthesis. Therefore, only topoisomerase II a may be regarded as a specific marker of cell proliferation. Although catalytic activity and protein content were not determined, our data are consistent with previous studies on the cell cycle-dependence of topoisomerase II isoforms in cultured cells [26,35]. The p170 enzyme increased during cell cycle progression in NIH-3T3 cells, while the p180 enzyme was constant once cells entered the cell cycle [35]. Moreover, the p180 isoform was present also in GO cells in contrast to the p170 isoform [35]. Altogether, these studies indicate that the two isoforms of topoisomerase II might perform different functions in cells. Detection of topoisomerase II content by polyclonal antibodies showed that the enzyme was not related to the proliferation state of Drosophila tissues during development [28]. As different forms of topoisomerase II have been shown to be present in Drosophila head tissues [27], investigations using specific antibodies against these isoforms may provide information on their different roles during cell proliferation. In our study, it was noteworthy that the a gene was expressed at higher levels than the/3 gene in the testis, which is a highly proliferating tissue. Since appreciable levels of topoisomerase II activity have been observed in termi-
nally differentiated chicken spermatids [30], one might speculate that the relatively high expression in the testis suggests that the p170 isoenzyme may be also involved in special functions during spermatogenesis. A major finding of the present study is the abrupt increase of topoisomerase II gene expression 1 day after birth in brain, liver and heart. A post-natal enhancement of D N A synthesis may not completely explain the increased expression, as this was not paralleled by a similar increase of thymidylate synthase gene expression. Moreover, the expression of the two topoisomerase II genes was differently regulated at early stages of development, as the expression of the/3 gene was specifically increased in the brain, while the a gene in the liver. These observations suggest that the two topoisomerase |I isoforms may be required during the development of specific differentiated tissues. Finally, as the two isoforms of topoisomerase II appear to be differentially inhibited by VM-26 and merbarone [34], the indication of a tissue-specific expression of them may have implications in a more rational clinical use of topoisomerase II-trapping agents. Conceivably, differences in gene expression could be exploited in the development of more selective topoisomerase II-targeted agents. Acknowledgements We wish to thank Dr. T.A. Dragani for helpful discussions. This work was supported in part by Consiglio Nazionale deile Ricerche 'Applicazioni Cliniche della Ricerca Oncologica'. References 1 Gellert, M. (1981) Annu. Rev. Biochem. 50, 879-910. 2 Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. 3 Gasser, S.M., Laroche, T., Falquet, J., Boy de la Tour, E. and Laemmli, U.K. (1986) J. Mol. Biol. 188, 613-629. 4 Earnshaw, W.C., Halligan, B., Cooke, C.A., Heck, M.M. and Liu, L.F. (1985) J. Cell Biol. 100, 1706-1715. 5 Earnshaw, W.C. and Heck, M.M. (1985) J. Cell Biol. 100, 17161725. 6 Berrios, M., Osheroff, N. and Fisher, P.A. (1985) Proc. Natl. Acad. Sci. USA 82, 4142-4146. 7 Mirkovitch, J., Mirault, M.E. and Laemmli, U.K. (1984) Cell 39, 223-232. 8 Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. 9 Pommier, Y. and Kohn, K.W. (1989) in Developments in Cancer Chemotherapy (Glazer, R.I., ed.), pp. 175-195, CRC Press, Boca Raton. 10 Capranico, G. and Zunino, F. (1990) in Molecular basis of specificity in nucleic acid-drug interactions (Pullman, B. and Jortner, J., eds.), pp. 167-175, Kluwer Academic Publishers, Netherlands. 11 Chow, K.C. and Ross, W.E. (1987) Mol. Cell. Biol. 7, 3119-3123. 12 Markovits, J., Pommier, Y., Kerrigan, D., Covey, J.M., Tilchen, E.J. and Kohn, K.W. (1987) Cancer Res. 47, 2050-2055. 13 Potmesil, M., Hsiang, Y.H.A.-L, Bank, B., Grossberg, H., Kir-
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14 15 16 17 18
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24 25 26 27
shenbaum, S., Forlenzar, T.J., Penziner, A., Kanganis, D., Knowles, D., Traganos, F. and Silber, R. (19881 Cancer Res. 48, 3537-3543. De Isabella, P., Capranico, G., Binaschi, M., Tinelli, S. and Zunino, F. (1990) Mol, Pharmacol. 37, 11-16. Davies, S.M., Robson, C.N., Davies, S.L. and Hickson, I.D. 11988) J. Biol. Chem. 263, 17724-17729. Sullivan, D.M., Latham, M.D. and Ross, W.E. (1987) Cancer Res. 47, 3973-3979. Giaccone, G., Gazdar, A.F., Beck, H., Zunino, F. and Capranico, G. (1992) Cancer Res. 52, 1666-1674. Fry~ A.M., Chresta, C.M., Davies, S.M., Walker, M.C., Harris, A.L., Hartley, J.A., Masters, J.R.W. and Hickson, I.D. (19911 Cancer Res. 51, 6592-6595. Duguet, M., Lavenot, C., Harper, F., Mirambeau, G. and De Recondo, A.M. (1983) Nucleic Acids Res. 11, 1059-1075. Taudou, G., Mirambeau, G., Lavenot, C., Dergarabedian, A., Vermeersch, J. and Duguet, M. (1984) FEBS Lett. 176, 431-435. Heck, M.M.S. and Earnshaw, W.C. 119861 J. Cell Biol. 103, 2569-2581. Heck, M.M.S., Hittelman, W.N. and Earnshaw, W.C. (1988) Proc. Natl. Acad. Sci. USA 85, 1086-1090. Woessner, R.D., Chung, T.D.Y., Hofmann, G.A., Mattern, M.R., Mirabelli, C.K., Drake, F.H. and Johnson, R.K. (1990) Cancer Res. 50, 2901-2908. Sullivan, D.M., Glisson, B.S., Hodges, P.K., Smallwood-Kentro, S. and Ross, W,E. (1986) Biochemistry 25, 2248-2256. Hsiang, Y.H., Wu, H.Y. and Liu, L.F. (1988) Cancer Res. 48, 32311-3235. Kaufmann, S.H., McLaughlin, S.J., Kastan, M.B., Liu, L.F., Karp, J.E. and Burke, P.J. (1991) Cancer Res. 51, 3534-3543. Hadlaczky, G., Praznovszky, T., Soft, J. and Udvardy, A. 11988) Nucleic Acids Res. 16, 10013-111023.
28 Whalen, A.M., McConnell, M. and Fisher, P.A. (1991) J. Cell Biol. 112, 203 213. 29 Fairman, R. and Brutlag, D.L. 11988) Biochemistry 27, 560-565. 30 Roca, J. and Mezquita, C. 11989) EMBO J. 8, 1855-1860. 31 Holden, J.A., Rolfson, D.H. and Wittwer, C.T. (19911) Biochemistry 29, 2127-2134. 32 Tsutsui, K., Sakurai, H., Shohmori, T. and Oda, T. (1986) Biochem. Biophys. Res. Commun. 138, 1116-1122. 33 Drake. F.H., Zimmerman, J.P., McCabe, F.L., Bartus, H.F., Per, S.R., Sullivan, D.M., Ross, W.E., Mattern, M.R., Johnson, R.K., Crooke, S.T. and Mirabelli, C.K. 11987) J. Biol. Chem. 262, 16739-16747. 34 Drake, F.tt., Hofmann, G.A., Bartus, |t.F., Mattern, M.R., Crooke, S.T. and Mirabelli, C.K. (19891 Biochemistry 28, 81548160. 35 Woessner, R.D., Mattern, M.R., Mirabelli, C.K., Johnson, R.K. and Drake, F.H. (19911 Cell Growth Diff. 2. 2119 214. 36 Chung, T.D.Y., Drake, F.H., Tan, K.B., Per, S.R., Crooke, S.T. and Mirabelli, C.K. 11989) Proc. Natl. Acad. Sci. USA 86, 94319435. 37 Austin, C.A. and Fisher M.L. (1990) FEBS Lett. 266, 115-117. 38 Tsai-Pflugfelder, M., Liu, L.F., Liu, A.A.. Tewey, K.M., WhangPeng, J., Knutsen, T., Huebner, K., Croce. C.M. and Wang, J.C. (1988) Proc. Natl. Acad. Sci. USA 85, 7177-7181. 39 Heller, D., Jackson, M. and Leinwand, k. (19841J. Mol. Biol. 173, 419-436. 40 Geyer, P.K. and Johnson, L.F. 119841 J. Biol. Chem. 259, 721)67211. 41 Sambrook, J., Fritsch. E.F. and Maniatis, T. 11989) Molecular cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 42 Jenh, C.H., Geyer, P.K. and Johnson, L.F. (1985) Mol. Cell. Biol. 5, 2527-2532.
43
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 92404
Different patterns of gene expression of top0isomerase II isoforms in differentiated tissues during murine development Giovanni Capranico, Stella Tinelli, Caroline A. Austin 1, Mark L. Fisher ' and Franco Zunino Dirision of Experimental Oncolo,~ B, lstituto Nazionale per lo Studio e la Cura dei Tumori, Milan (Italy)
(Received 16 March 1992)
Key words: Topoisomerase isoform; Gene expression; Normal tissue; Differentiation; Development; (Mouse) The expression of DNA topoisomerase II a and /3 genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II a gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II /3 gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the /3 gene expression was observed in the brain but not in the liver; conversely, the expression of the a gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II a may be a specific marker of cell proliferation, whereas topoisomerase II /3 may be implicated in functions of DNA metabolism other than replication.
Introduction D N A topoisomerases II are essential enzymes in living cells playing a role in vital processes, such as D N A replication, transcription, recombination and repair [1,2]. In addition, eukaryotic topoisomerase II is a structural c o m p o n e n t of mitotic c h r o m o s o m e scaffolds [3-5] and the nuclear matrix of interphase cells [6] and has been implicated in sister chromatid exchange [1,2] and in the organisation of D N A loop domains [7]. The nuclear topoisomerase II is a major target of clinically effective antineoplastic drugs [8-10]. These agents reversibly stabilize a normal intermediate formed during the enzyme-catalyzed reaction, resulting in D N A cleavage stimulation. T h e drug action then initiates a complex and poorly understood cascade of events culminat-
Correspondence to: G. Capranico, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. i Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, London, UK.
ing in cell death [1,2,8,9]. Although many factors play a role in the drug cytotoxic activity, one important determinant is the cellular content of topoisomerase II [11-18]. The a m o u n t and activity of topoisomerase II were shown to correlate with the state of cell proliferation in normal cells [19-22], missing in nonproliferative tissues and quiescent cells. The regulation of topoisomerase II levels in t u m o r cells seems to be more complex. Topoisomerase II levels have been reported to be higher in transformed than in normal cells, and not to decline to low levels when cancer cells cease proliferation [23-25]. Thus, topoisomerase II regulation may be altered in transformed as o p p o s e d to normal cells, representing a basis for the anti-tumor activity of topoisomerase II inhibitors. However, topoisomerase II levels in highly proliferating h u m a n hematopoietic progenitor cells were shown to be indistinguishable from levels observed in h u m a n leukemia cell lines [26] and markedly decreased both in m a t u r e hematopoietic cells and in leukemic cells induced to differentiate [26]. O n the other hand, appreciable amounts of topoisomerase II
44 have been described in non-proliferating Drosophila normal tissues [27-29], chicken late spermatids [30], some normal human tissues [31] and in rat neuronal nuclei [32]. Therefore, these observations documented that topoisomerase lI may be present in some terminally differentiated cells, although detailed information is missing on the regulation of topoisomerase II in normal tissues during differentiation. In recent years, human and murine tumor cells have been shown to contain two isoenzymes of topoisomerase II, termed p170 and p180, which have been reported to be pharmacologically and biochemically distinct [33,34]. The two isoforms might be differentially regulated during changes in the proliferation state [35], in ras-trasformed murine cells [23] and in drug-resistant tumor cells [33]. Non-overlapping sets of topoisomerase lI c D N A fragments were recently cloned from human cells, and shown to derive from two distinct genes, a and /3, which likely encode for the two isoforms p170 and p180 of topoisomerase II, respectively [36-38]. Many of the previous investigations on topoisomerase II content in mammalian cells have been performed with western blotting techniques often using antibodies specific for the p170 isoform. The availability of specific D N A probes for the two topoisomerase I1 genes, then, prompted us to investigate the expression of these g e n e s in normal murine tissues in the attempt to understand the functions of D N A topoisomerase II isoforms in normal differentiated cells. The expression of the two genes was examined in thirty normal tissues of adult and newborn mice, and in murine embryos.
Materials and M e t h o d s
cDNA probes As murine topoisomerase II isoform gene probes were not available, human topoisomerase II e D N A fragments were used in this study. Two different e D N A fragments were used to detect specifically the topoisomerase II ~ gene (coding for the p170 form): the h-Top2-Z2 probe [38], corresponding to a 3' terminal portion of the gene, provided by Dr. L.F. Liu; and the p G T O P 2 - 1 probe, corresponding to a 5' terminal portion of the gene, recently cloned from the human NCI-H187 cell line using a PCR-based procedure [17]. The c D N A clone CAA5, obtained from a H e L a cell c D N A library [37], was used to specifically determine the expression of the topoisomerase II /3 gene (coding for the p180 form). As a control probe for RNA loading, a mouse LLRep3 0.8 kb Pst I D N A fragment was derived from plasmid pLLRep3 [39]. The mouse thymidylate synthase probe, a 1 kb Pst I fragment, was derived from plasmid pMTS-3 [40]. RNA extraction and Northern blot analysis C3H mice were purchased from Charles River (Calco, Italy). Mice were maintained and treated following the Principles and Guidelines for the Use of Animals in Research of the Istituto Nazionale per lo Studio e la Cura dei Tumori. Tissues were excised from 3-month-old healthy mice (adult animals) or 1-day-old mice (newborns) and cleaned of surrounding material. Embryos were excised 16, 8 or 2 days before delivery and cleaned of parental tissues. Total R N A was immediately extracted by the LiCl-guanidine monothio-
#.,o
6.2
K b -,,.
6.2
K b .-,-
"4" . . . .
""
_
.o
,r
.........
•
Topo
II 0
T O p O II I~'
Fig. 1. Northern blot analysis of topoisomerase II a and /3 gene expression in tissues of adult mice. Total RNAs were fractionated on a denaturing 1% agarose gel, transferred to nylon membranes and hybridised with the h-Top2-Z2 (upper panels) or CAA5 (lower panels) probes in 50% formamide, 5 x SSC, 0.2% SDS, 5 x Denhardt's solution, 50 mM sodium phosphate (pH 7), 250/zg/ml of salmon sperm DNA, 10% dextran sulphate at 42°C for 20 h. Final washes of filters were at 55°C in 0.I XSSC, which did not result in a cross-hybridization between the a and /3 probes.
45
cyanate method [41]. RNA (20/zg) was fractionated on a formaldehyde-containing 1% agarose gel and then transferred to a nylon membrane (Hybond). The membrane was irradiated with ultraviolet light and prehybridization was for at least 4 h at 42°C in 50% formamide, 5 x SSC, 0.2% sodium dodecyl sulphate (SDS), 5 × Denhardt's solution, 50 mM sodium phosphate (pH 7), 250 tzg/ml of salmon sperm DNA. DNA probes were labelled at a specific activity of 2 to 5 - 10 ~ cpm/Izg DNA with the Multiprime DNA labelling systems and deoxycytidine-5'[a- 32P]triphosphate (3000 Ci mmol -~) (Amersham International, UK). Hybridization was carried out for 20 h at 42°C in the same buffer containing 10% dextran sulphate. Final wash of filters was at 55°C in 0.1 x SSC. Autoradiography was at -70°C for 1 to 7 days.
A
e^ 1
2
n 3
4
a 5
6
e
n
7
8
6.2 Kb.~i
To
. p
6.2
Topo II a
Results
Expression of topoisomerase H isoform genes in adult mice Total RNA was purified from each of 22 normal tissues of 3-month-old mice and examined for topoisomerase II a and /3 gene expression by Northern blots using the h-Top2-Z2 [38] and CAA5 probes [37] (Figs. 1 and 2). These probes detected m R N A species of 6.2 kb in all the tissues studied. The patterns of expression differed markedly among examined tissues, showing that the cDNA probes used specifically recognized distinct transcripts in murine RNA. The a gene was highly expressed in the thymus (Fig. 2A, lane 5; B, lane 8), spleen and marrow (Figs. 1 and 2), and to a lesser extent in the intestine, stomach and testis (Fig. 1). Northern blotting analysis using a second topoisomerase II a probe, pGTOP2-1 [17], gave the same results (not shown). The /3 gene was expressed more evenly and in a wider range of the studied tissues of adult mice (Figs. 1 and 2). In particular, a relatively high expression was observed not only in the spleen and marrow but also in the uterus, ovary, lymph nodes, adrenal gland, eye, bladder and heart (Fig. 1). The expression level of the two genes in each mouse tissue was determined relative to the expression level in the thymus (Table I), this being the tissue in adult mice expressing both genes at the highest levels (Fig. 2). RNA loading on gels was checked by ethidium bromide staining and densitometry of gel photographs. Re-hybridizations of filters with a control DNA probe, LLRep3, was also performed in some cases (not shown). Tissues were ranked in four groups with undetectable, low, intermediate and high expression levels (Table I). The patterns of gene expression were markedly different for the two isoenzymes, with the a gene being expressed in fewer tissues than the /3 gene (5 and 15 tissues, respectively, at 'intermediate' and 'high' levels, Table I). Expression of the a gene in the 'low' group
B
n
a
1 2345678
6.2 Kb'~
Topo II 0
Fig. 2. Topoisomerase II a and /3 gene expression in tissues from newborn mice, embryos and selected tissues of adults. See legend to Fig. 1 for methods. Letters e, n and a indicate embryos, newborns and adults, respectively. (A) Lanes 1-3, total embryos 16, 8 and 2 days before delivery, respectively; 4, lung of newborns; 5, thymus of adults; 6, spleen of adults; 7, heart of embryos 2 days before delivery; 8, heart of newborns. (B) Lanes 1, lung; 2, thymus; 3, heart; 4, breast; 5, ovary; 6, marrow; 7, testis; 8, thymus.
(Table I) was detected on autoradiograms by using longer exposure times than those shown in Figs. 1-3. Thus, topoisomerase II a was expressed mainly in proliferative tissues, while topoisomerase II /3 was also expressed at intermediate levels in many non-proliferating tissues. Of tissues examined in adult mice, only the pancreas, salivary glands and muscle did not show detectable expression of either gene (Table I).
Topoisomerase H gene expression in murine embryos and newborns The a and /3 gene expressions were also examined during murine embryogenesis and in newborns (1-dayold mice), and compared to the thymus and other selected tissues of adult mice. Expression of the/3 gene in whole embryos at 16 days before delivery was higher (2.5-fold) than in the thymus of adult mice, and dropped to low levels in embryos 8 and 2 days before delivery (Fig. 2A, lanes 1-3 and 5). High levels of the a gene expression were also seen in early embryos which dropped in older embryos (Fig. 2A); however, it was
46 TABLE 1
BRAIN
Expression o f topoisomerase 11 a and fl genes in dif]~'rentiated murine tissues
A
r
1
LIVER "I
2
3
r
4
A
5
6
Expression level " undetectable
low
Topoisomerase ll c~ gene kidney, liver, breast, ~ muscle, brain, stomach, lung, pancreas, ()vary, uterus, bladder, heart, lymph nodes, eyes, tongue, saliwlry glands, adrenal glands. Topoisomerase 11 fl genc pancreas, liver, salivary glands, testis, muscle, kidney, brain,
intermediate
high
0.6 intestine, (I.4 testis, 0.4 0.3
2.2 thymus, 1.5 marrow, spleen,
10 4.7 3.9
0.3 0.3 0.2 0.1
2.9 thymus, 2.6 2.5 2.3 2.0 1.9 1.6 1.4 1.3 1.1 1.1 0.9 0.9 0.9
10
adrenal glands. ovary, spleen, lymph nodes, uterus, heart, eyes, marrow, bladder, tongue, lung, stomach, intestine, breast, ~'
" Levels of topoisomerase II gene expression are shown relative to those of thymus, set equal to 10. The data shown were calculated from 2 to 3 independent experiments. h From female mice I day after delivery.
never higher than that in the thymus of adult mice, the tissue with the highest level of a gene expression. The decrease in expression in older embryos could be partly due to a dilution effect, as both genes were expressed in tissues of embryos 2 days before delivery at detectable levels (Fig. 2A, lane 7; Fig. 3, lanes 1 and 4). The thymus, lung, liver, brain and heart were evaluated in 1-day-old mice, and expression of both genes was observed in all these tissues (Figs. 2 and 3). Again, the expression levels of the a and /3 genes were different in the newborn tissues (Table If). The thymus and liver expressed the highest levels of a topoisomerase lI RNA and the brain the lowest one, while levels of /3 gene expression were highest in the brain and lowest in the liver.
Post-natal changes of topoisomerase H gene expression Topoisomerase isoform expression was further compared in the liver and brain during murine development. The a gene was expressed in these tissues from embryos (2 days before delivery) and newborns, but not from adult mice (Fig. 3). An increase in gene expression for this isoform was observed in the newborn
6.2 Kb .-~
Topo II a
6.2 Kb -~
Topo In 13
1.3 K b ~
TS t
Fig. 3. Analysis of topoisomerase 11 ce and fl gene expression in murine brain and liver during development. See legend of Fig. 1 for methods. The filters were further rehybridised with a mouse D N A probe for the thymidylate synthase gene (TS). Lanes 1 and 4, embryos 2 days before delivery: 2 and 5, newborns: 3 and 6, adults.
tissues as compared to the embryo tissues, with a prominent increase in the liver, about 2 to 3-fold (Fig. 3, upper panel). The liver and brain of adult mice expressed low levels of topoisomerase II /3 (Table I); strikingly, a marked increase (more than 6-fold) of expression of the /3 gene was observed in the brain of T A B L E 11 Expression of topoisomerase 11 a and fi genes in tissues ~f newborn mice " Tissue
o~ gene
13 gene
Thymus (adults) Thymus Liver Heart Lung Brain
10 4.1 3.6 1.9 1.6 1.5
10 8.8 2.6 8.2 9.8 18.1
~' l-day-old mice. Levels of topoisomerase II gene expression are shown relative to those of adult mouse thymus, set equal to 10. The data shown were calculated from 2 to 3 independent experiments.
47 newborns as compared to the embryos, but only a slight increase was observed in the case of the liver (Fig. 3, middle panel). These increases in the postnatal period were not correlated with an increase in the cell division rate as evaluated by the expression level of the thymidylate synthase gene (Fig. 3, lower panel), a marker of cell proliferation [42]. For the /3 gene, a similar increase in expression was also observed in the heart of newborn mice as compared to that of embryos (Fig. 2A, lanes 7 and 8). Discussion The present results indicate that topoisomerase II and /3 genes are differentially expressed in normal murine tissues. Among 22 tissues from adult (3-monthold) mice, the a gene expression was essentially restricted to five tissues, while the/3 gene was expressed in 15 tissues at high or intermediate levels (Table I). The thymus was the tissue that expressed both genes at the highest level in adult mice. Moreover, in several tissues only the /3 gene expression was detectable (heart, adrenal glands, lymph nodes, uterus, eyes, ovary, bladder, tongue and lung). The c~ gene was expressed in tissues characterized by a relatively high fraction of replicating cells, suggesting a main role of the p170 isoenzyme in functions associated with D N A replication. In contrast, the /3 gene was also expressed in tissues not expected to have proliferating cells, suggesting that the /3 isoform may also participate in D N A metabolic processes other than D N A synthesis. Therefore, only topoisomerase II a may be regarded as a specific marker of cell proliferation. Although catalytic activity and protein content were not determined, our data are consistent with previous studies on the cell cycle-dependence of topoisomerase II isoforms in cultured cells [26,35]. The p170 enzyme increased during cell cycle progression in NIH-3T3 cells, while the p180 enzyme was constant once cells entered the cell cycle [35]. Moreover, the p180 isoform was present also in GO cells in contrast to the p170 isoform [35]. Altogether, these studies indicate that the two isoforms of topoisomerase II might perform different functions in cells. Detection of topoisomerase II content by polyclonal antibodies showed that the enzyme was not related to the proliferation state of Drosophila tissues during development [28]. As different forms of topoisomerase II have been shown to be present in Drosophila head tissues [27], investigations using specific antibodies against these isoforms may provide information on their different roles during cell proliferation. In our study, it was noteworthy that the a gene was expressed at higher levels than the/3 gene in the testis, which is a highly proliferating tissue. Since appreciable levels of topoisomerase II activity have been observed in termi-
nally differentiated chicken spermatids [30], one might speculate that the relatively high expression in the testis suggests that the p170 isoenzyme may be also involved in special functions during spermatogenesis. A major finding of the present study is the abrupt increase of topoisomerase II gene expression 1 day after birth in brain, liver and heart. A post-natal enhancement of D N A synthesis may not completely explain the increased expression, as this was not paralleled by a similar increase of thymidylate synthase gene expression. Moreover, the expression of the two topoisomerase II genes was differently regulated at early stages of development, as the expression of the/3 gene was specifically increased in the brain, while the a gene in the liver. These observations suggest that the two topoisomerase |I isoforms may be required during the development of specific differentiated tissues. Finally, as the two isoforms of topoisomerase II appear to be differentially inhibited by VM-26 and merbarone [34], the indication of a tissue-specific expression of them may have implications in a more rational clinical use of topoisomerase II-trapping agents. Conceivably, differences in gene expression could be exploited in the development of more selective topoisomerase II-targeted agents. Acknowledgements We wish to thank Dr. T.A. Dragani for helpful discussions. This work was supported in part by Consiglio Nazionale deile Ricerche 'Applicazioni Cliniche della Ricerca Oncologica'. References 1 Gellert, M. (1981) Annu. Rev. Biochem. 50, 879-910. 2 Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. 3 Gasser, S.M., Laroche, T., Falquet, J., Boy de la Tour, E. and Laemmli, U.K. (1986) J. Mol. Biol. 188, 613-629. 4 Earnshaw, W.C., Halligan, B., Cooke, C.A., Heck, M.M. and Liu, L.F. (1985) J. Cell Biol. 100, 1706-1715. 5 Earnshaw, W.C. and Heck, M.M. (1985) J. Cell Biol. 100, 17161725. 6 Berrios, M., Osheroff, N. and Fisher, P.A. (1985) Proc. Natl. Acad. Sci. USA 82, 4142-4146. 7 Mirkovitch, J., Mirault, M.E. and Laemmli, U.K. (1984) Cell 39, 223-232. 8 Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. 9 Pommier, Y. and Kohn, K.W. (1989) in Developments in Cancer Chemotherapy (Glazer, R.I., ed.), pp. 175-195, CRC Press, Boca Raton. 10 Capranico, G. and Zunino, F. (1990) in Molecular basis of specificity in nucleic acid-drug interactions (Pullman, B. and Jortner, J., eds.), pp. 167-175, Kluwer Academic Publishers, Netherlands. 11 Chow, K.C. and Ross, W.E. (1987) Mol. Cell. Biol. 7, 3119-3123. 12 Markovits, J., Pommier, Y., Kerrigan, D., Covey, J.M., Tilchen, E.J. and Kohn, K.W. (1987) Cancer Res. 47, 2050-2055. 13 Potmesil, M., Hsiang, Y.H.A.-L, Bank, B., Grossberg, H., Kir-
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