Involvement of DNA Topoisomerase IIbeta in Neuronal Differentiation*

Ken TsutsuiDagger §, Kimiko Tsutsui, Kuniaki Sano, Akihiko Kikuchi||, and Akira Tokunaga

From the Dagger  Department of Molecular Biology, Institute of Cellular and Molecular Biology, the  Third Department of Anatomy, Okayama University Medical School, Okayama 700-8558, Japan and the|| Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan

Received for publication, September 18, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells. While topo IIalpha is essential for chromosome segregation in mitotic cells, in vivo function of topo IIbeta remains to be clarified. Here we demonstrate that the nucleoplasmic topo IIbeta , highly expressed in differentiating cerebellar neurons, is the catalytically competent entity operating directly on chromatin DNA in vivo. When the cells reached terminal differentiation, this in vivo activity decreased to a negligible level with concomitant loss of the nucleoplasmic enzyme. Effects of topo II-specific inhibitors were analyzed in a primary culture of cerebellar granule neurons that can mimic the in vivo situation. Only the beta  isoform was expressed in granule cells differentiating in vitro. ICRF-193, a catalytic topo II inhibitor, suppressed the transcriptional induction of amphiphysin I which is essential for mature neuronal activity. The effect decreased significantly as the cells differentiate. Expression profiling with a cDNA macroarray showed that 18% of detectable transcripts were up-regulated during the differentiation and one-third of them were susceptible to ICRF-193. The results suggest that topo IIbeta is involved in an early stage of granule cell differentiation by potentiating inducible neuronal genes to become transcribable probably through alterations in higher order chromatin structure.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA topoisomerase II (topo II)1 is essential for cell proliferation since some mitotic events such as condensation and segregation of daughter chromosomes are entirely dependent on its activity. It is also likely to be involved in other DNA transactions like DNA replication, transcription, and recombination (1). The enzyme, in a heart-shaped dimer form (2), catalyzes topological transformations of DNA by transient cleavage of DNA double strands forming an enzyme-operated gate, followed by passing a second double-stranded DNA segment through the gate (3, 4). Only one form of topo II is present in lower eukaryotes and invertebrates whereas in mammalian cells two isoforms, the 170-kDa topo IIalpha and the 180-kDa topo IIbeta , that are encoded by separate genes have been identified (5-7). These isoforms may share their roles in different cellular functions although they show similar catalytic properties in vitro. From the following observations, topo IIalpha is considered to be the main isoform involved in the mitotic processes. First, there is a positive correlation between the cellular concentration of topo IIalpha and the rate of cell proliferation (8, 9). Second, the expression of mRNA for topo IIalpha is higher in tissues containing proliferating cells (10) and is localized in brain regions enriched with mitotic cells (11, 12). Third, the level of topo IIalpha protein peaks at G2/M phase during the cell cycle (9, 13) and finally, high affinity binding of topo IIalpha with chromatin at mitosis is essential for chromosome condensation/segregation and topo IIbeta cannot be substituted for topo IIalpha (14).

In contrast to topo IIalpha , functional aspect of topo IIbeta remains obscure (1, 15). Our previous studies demonstrated a significant level of topo II activity in isolated nuclei from post-mitotic neuronal cells (16, 17), that was shown later to be attributable to the beta  isoform (11). Expression of topo IIbeta was also shown in other nonproliferative or fully differentiated tissues at both the transcript (10) and the protein levels (18, 19). Cell cycle analysis in cultured cells showed that the level of topo IIbeta was not altered significantly and, in a sharp contrast to topo IIalpha , it rather increased slightly as cells reached a plateau phase (9, 13, 20). Expression of topo IIbeta in these immortalized cell lines, however, is probably deregulated. Topo IIbeta is even dispensable for cell proliferation and survival in vitro since in some cell lines the enzyme is not expressed at all (21). These observations suggest that topo IIbeta is not required for maintenance of general cellular activities but rather involved in more specific processes in vivo.

The cerebellar cortex has a unique structure with laminar arrangement of cells that is formed during the development in early postnatal period. A number of mRNAs were identified to be expressed specifically at distinct stages of cerebellar development (22, 23). We have shown previously using in situ hybridization that in the first two postnatal weeks the signal levels for topo IIbeta mRNA increased on the cerebellar granule cells actively differentiating in the granular layer, suggesting a link between the topo IIbeta expression and the cerebellar development (12). The cerebellar system also appears to be ideal in elucidating the physiological function of topo IIbeta since the control of its expression in cultured cell lines may well be aberrant. Parenthetically, there have been no detailed studies on temporo-spatial expression of topo II isoforms in living tissues. In the present study, we discriminated the catalytically competent entity of topo II isoforms that is acting on chromatin DNA in the developing cerebellar tissue. Postnatal expression levels and intranuclear localization of topo IIbeta were also traced in the cerebellar neuronal lineages, Purkinje cells and granule cells. We finally assessed the requirement of topo IIbeta activity in the expression of neuronal genes in a primary culture of differentiating granule cells by use of topo II-specific inhibitors. Our results are consistent with the defects in embryonic neural development identified recently in topo IIbeta knockout mice (24).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies

Topoisomerase IIalpha -- Full-length cDNA for human topo IIalpha was expressed in Escherichia coli, and mice were immunized with purified protein to prepare monoclonal antibodies as described previously (25). One of the isolated clones, 4E12, recognized a NH2-terminal fragment (residues numbers 1-40), and was cross-reactive to rodent topo IIalpha (26).

Topoisomerase IIbeta -- Full-length cDNA for human topo IIbeta was expressed and mouse monoclonal antibodies were prepared as above. One of the isolated clones, 3B6, was also reactive to rodent topo IIbeta (26), recognizing a COOH-terminal fragment (residues numbers 1178-1273).

Amphiphysin I-- IgG was purified from the serum of a breast cancer patient with anti-amphiphysin I autoantibodies and paraneoplastic sensory neuropathy (27).

Neurofilament-- A mouse monoclonal antibody to the rat neurofilament proteins (160 K/210 K) was purchased from IBL (Gunma, Japan).

Western Blot Analysis

Nuclei were prepared from cerebellum, cerebral cortex, and thymus of male Wistar rats as described previously (28). Nuclear samples were pretreated with 50 µg/ml DNase I at room temperature for 30 min prior to electrophoresis. Topo II-enriched fraction was extracted from the purified nuclei with 20 mM Tris-HCl (pH 7.5), 0.3 M NaCl, and 140 mM 2-mercaptoethanol. Cultured granule cells were directly lyzed in the loading buffer with a brief sonication. Samples were heated at 95 °C for 1 min in a loading buffer and then subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins in gels were transferred to nitrocellulose membranes by electroblotting and processed for immunodetection according to a standard procedure. Secondary antibodies were peroxidase-conjugated goat antibodies (Bio-Rad) and the peroxidase activity was visualized with 4-methoxy-1-naphthol and hydrogen peroxide.

Immunohistochemical Detection

Male Wistar rats under deep anesthesia were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde and 0.2% picric acid in 100 mM sodium phosphate (pH 7.4). The brain was dissected out, post-fixed with the same solution, and cryoprotected with 20% sucrose in 100 mM sodium phosphate (pH 7.4). After trimming, the brain blocks were frozen rapidly and sectioned at a thickness of 10 µm on a cryostat.

Frozen sagittal sections were thaw-mounted on glass slides precoated with 3-aminopropyltriethoxysilane, followed by washing with PBS (150 mM NaCl in 10 mM sodium phosphate, pH 7.4), blocking with PBS containing 10% goat serum and 0.3% Triton X-100, and incubation for 15 h with primary antibodies diluted in PBS containing 0.3% Triton X-100 (TPBS). The incubation was performed either at 37 °C (monoclonal antibodies against topo II isoforms) or at room temperature (all the other antibodies). After washing with TPBS, the sections were incubated successively at room temperature with biotinylated goat secondary antibodies (Vector Labs) and with the ABC reagent (Vectastain ABC Kit, Vector Labs). The peroxidase reaction was visualized with diaminobenzidine and hydrogen peroxide.

Cultured granule cells that were grown on coverslips precoated with poly-L-lysine were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. The fixed cells were immunostained as described above for tissue sections.

Immunofluorescence Microscopy and Confocal Microscopy

The brain tissue was perfused as described above except that the solution did not contain picric acid. Frozen sagittal sections (14 µm) were mounted on glass slides as mentioned, washed with PBS, permeabilized with 0.3% Triton X-100 in PBS, and blocked with 10% goat serum and 10% horse serum in TPBS. After incubation with various primary antibodies diluted in TPBS containing 3% horse serum for 15 h, sections were washed with PBS, followed by incubation with fluorescent conjugates of goat secondary antibodies in PBS at room temperature for 30 min. DNA was then stained with 0.25 µg/ml 4',6-diamidino-2-phenylindole in PBS. Stained sections were mounted in an antifade solution (Prolong Antifade Kit, Molecular Probes, Eugene, OR) and examined under an OLYMPUS epifluorescence microscope (BX60-FLB) coupled to a cooled monochromatic CCD camera (Hamamatsu Photonics) or OLYMPUS laser scanning microscope (FLUOVIEW-BX50). Recorded digital images were processed later using Adobe Photoshop software.

Determination of Topoisomerase Activities in Vivo

From male Wistar rats of P10, the cerebellum was dissected out under deep anesthesia and immediately cut into 1-mm thick slices on ice (about 10 sagittal slices), submerged in 1 ml of RPMI 1640 with or without 100 µM etoposide, and incubated at 37 °C for 2 h in a CO2 incubator. The slices were homogenized with 3 ml of 1% (v/v) Sarkosyl in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA (TE buffer), and then fractionated by centrifugation on a CsCl step gradient (2-ml layers of CsCl solutions in TE buffer with densities of 1.82, 1.72, 1.50, and 1.37 g/cm3) according to Muller and Mehta (29). Density of the gradient fractions (0.5 ml) was calculated from the refractive index of each fraction. DNA concentration was estimated by a spectrophotometric method based on the Warburg-Christian equation. Aliquots (47 µl for topo IIalpha detection and 12 µl for topo IIbeta detection) of each fraction were diluted 1:3 with TE buffer and blotted onto nitrocellulose membranes using a slot blotting apparatus (Bio-Dot SF, Bio-Rad). The membranes were then processed for immunostaining with topo II isoform-specific monoclonal antibodies according to the procedure described above. Stains on the membrane were scanned on a scanner and topo II isoforms in each fraction were quantified using a densitometric software, Intelligent Quantifier (Bio Image).

Primary Culture

Cerebellar granule cells were prepared from the cerebellum of P8 Wistar rats and processed for culture as described previously (30). The tissue was treated briefly with trypsin and DNase I and the dispersed cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 25 mM KCl, and 60 µg/ml kanamycin sulfate. The cell suspension from one cerebellum was dispensed into 6 plastic culture dishes (60 mm in diameter) precoated with poly-L-lysine. After 24 h in culture, proliferation of non-neuronal cells was prevented by replacing the medium with that containing 10 µM cytosine arabinoside. The medium was refreshed at the end of day 3. Cells remained viable under these conditions for at least 8 days in culture.

Northern Blot Analysis

Total cellular RNA was prepared from the cultured cells by using a commercial kit (RNeasy, Quiagen). RNA samples (17 µg/lane) were electrophoresed and blotted as described previously (11). cDNA clones encoding the neuronal markers (amphiphysin I, amphiphysin II, synaptophysin, and gamma -aminobutyric acid receptor alpha 6) were first obtained by polymerase chain reaction amplification of a rat brain cDNA library (Uni-Zap XR, Stratagene) with unique primer sets designed to amplify 500-1500-base pair regions of the cDNAs. Products were cloned into the pGEM-T vector (Promega) and verified by nucleotide sequencing. Hybridization probes were prepared by polymerase chain reaction amplification of the cloned cDNA fragments with the same primer sets. The purified products were labeled by a random-primed synthesis with [alpha -32P]dCTP. Hybridization was carried out under standard conditions at 42 °C overnight in a solution containing 50% (v/v) formamide.

mRNA Expression Profiling

A cDNA macroarray system (Atlas Rat 1.2 Array, Clontec) was used for determination of relative levels of mRNAs that are expressed in granule neurons differentiating in vitro. Total cellular RNA was isolated from untreated granule cells at day 1, at day 5, and ICRF-treated cells at day 5 as described above. The following procedures were performed according to the protocol supplied by the manufacturer. Briefly, RNA preparations were first treated with DNase I, followed by enrichment of poly(A)+ RNA on streptoavidin magnetic beads. RNA templates bound to the beads were subjected to cDNA synthesis with rat CDS primer mixture and [alpha -32P]dATP. Purified cDNA probes (>6.5 × 106 cpm) were hybridized to the arrayed rat cDNA membranes under recommended conditions. Washed membranes were exposed against the imaging plate for 12 h at room temperature and hybridization signals were quantified on the BAS 2000 analyzer using an array-analysis software ArrayGauge (Fuji Film).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of DNA Topoisomerase II Isoforms in Rat Cerebellar Cortex at Postnatal Day 10 (P10)-- As we have shown previously, mRNAs of both topo IIalpha and topo IIbeta were detected by in situ hybridization in the P10 rat cerebellum (11). To confirm the expression of these enzymes at the protein level, nuclei isolated from rat tissues were first subjected to Western blot analysis. A monoclonal antibody, 3B6, raised against human topo IIbeta specifically recognized a 180-kDa polypeptide in rat cerebellum (Fig. 1, lane 5). A band with the same mobility was also detected in cerebral cortex (lane 6) but not in P14 rat thymus (lane 4) unless the color development was prolonged. In contrast to topo IIbeta , Western blot signals were quite low when total nuclear proteins were probed with a monoclonal antibody, 4E12, raised against human topo IIalpha . However, when nuclear salt extracts enriched with topo II were used for the analysis, the antibody detected an immunoreactive band of 170 kDa, the molecular mass expected for canonical topo IIalpha , in both thymus and cerebellum (Fig. 1, lanes 1 and 2), but still failed to detect any reactive bands in cerebral cortex (lane 3). These results are consistent with the levels of transcripts estimated previously by Northern blot analysis and further support the idea that the expression of topo IIalpha is tightly associated with cell proliferation whereas that of topo IIbeta is not (11). These results also confirmed that both isoforms of topo II are expressed in P10 rat cerebellum and that the monoclonal antibodies, 4E12 and 3B6, can be used as a specific probe for topo IIalpha and topo IIbeta , respectively.



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Fig. 1.   Specific detection of topo II isoforms in rat nuclei by immunoblotting. Nuclear proteins extracted with 0.3 M NaCl and 140 mM 2-mercaptoethanol (lanes 1-3, 30 µg of protein/lane) or whole nuclei treated with DNase I (lanes 4-6, 5 µg of protein/lane), were subjected to 6% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. Blotted membranes were probed with mouse monoclonal antibodies raised against human topo IIalpha , 4E12 (lanes 1-3, 2 µg of IgG/ml) or human topo IIbeta , 3B6 (lanes 4-6, 1 µg of IgG/ml). Lanes 1 and 4, P14 rat thymus nuclei; lanes 2 and 5, P10 rat cerebellar nuclei; lanes 3 and 6, P10 rat cerebral cortex nuclei; lane M, molecular weight markers.

Catalytic Activities of DNA Topoisomerase II Isoforms Detected in Vivo during the Postnatal Development of the Cerebellum-- The results presented above or simple immunohistochemical detection methods with specific antibodies do not provide information on the abundance of catalytically active topo II molecules. Although it is possible to measure topo II activities in tissue extracts, this in vitro activity on naked DNA does not reflect the topo II enzyme acting on DNA in situ, since their accessibility is largely restricted in vivo by local chromatin structures (31, 32). The way to circumvent this problem is to take advantage of the unique property of topo II enzymes. A class of topo II inhibitors, sometimes referred to as "topo II poisons," such as etoposide (VP-16) binds specifically to the enzyme and stabilizes the topo II-DNA covalent complex (cleavable complex) by preventing the religation of transiently cleaved DNA strands (33). Upon addition of strong detergents, the complex is disrupted to liberate DNA fragments with denatured topo II protein covalently attached to the 5' termini. For quantitative estimation of functional topo II isoforms in the developing rat cerebellum, cerebellar slices were incubated with etoposide as described under "Experimental Procedures." The tissue was then homogenized in Sarkosyl, and the functioning topo II molecules cross-linked to DNA were separated from the free enzyme by CsCl step gradient centrifugation. Gradient fractions were blotted onto membranes and probed with the monoclonal antibodies to topo IIalpha or topo IIbeta , thus enabling the isoform-specific detection.

Fig. 2 shows a representative result obtained with P10 rat cerebellum. When the tissue was incubated in the absence of etoposide, both topo IIalpha and topo IIbeta were completely recovered in fractions with a density of typical proteins (Fig. 2, A and C). In the presence of etoposide, however, 55% of topo IIalpha and 22% of topo IIbeta were recovered in the DNA peak fractions at around 1.69 g/cm3 (Fig. 2, B and D). The immunoreactive material in the DNA peak fractions represents the topo II-DNA complexes accumulated during the incubation with etoposide, and thus reflects an in vivo activity of enzymes that are accessible to chromatin DNA. The density of these complexes is comparable to that of bulk DNA because of the small topo II versus DNA mass ratio. When the lysate DNA was fragmented by sonication before loading the gradient, the peak of topo II-DNA complexes shifted toward the right depending on the extent of sonication and became separated from the DNA peak. By estimating the average length of DNA fragments, it was calculated that under these conditions one or two topo II monomers are cross-linked to a few kilobase pairs of DNA.



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Fig. 2.   Fractionation of topo II-DNA covalent complexes induced by etoposide. P10 rat cerebellar slices were incubated with (B and D) or without (A and C) 100 µM etoposide, lysed, and fractionated by CsCl density gradient centrifugation as described under "Experimental Procedures." Aliquots from the fractions were blotted onto nitrocellulose membranes and the topo II isoforms were detected separately with the isoform-specific monoclonal antibodies (3 µg/ml). After scanning the stained membranes, topo II isoforms were quantified by densitometry. A and B, topo IIalpha ; C and D, topo IIbeta . Open circles, amounts of topo II in densitometric units; closed circles, DNA concentrations. Fraction densities were determined by refractometry and shown here by broken lines.

In rodents, the gross organization of cerebellar tissue initiates after birth and is completed within 3 weeks. During this period, the precursor for granule cells, the most abundant neurons in central nervous system, proliferate in the outmost layer (external germinal layer, EGL), then migrate inward through the molecular layer and the Purkinje cell layer to form the granular layer, where they continue to differentiate establishing neuronal connections (34). Using the cross-linking technique, we next assessed the in vivo activity of topo II isoforms and their total protein levels during the postnatal development of rat cerebellum (Fig. 3). The difference in the densitometric levels indicated on the ordinate roughly reflects the relative abundance of topo II isoforms in the cerebellum. Topo IIalpha linked to DNA increased rapidly during the first 5 days after birth, reaching a peak at P9, and sharply decreased thereafter in the second week to a negligible level at P20 (Fig. 3A). More than 50% of topo IIalpha was cross-linked to DNA between P5 and P10 when its expression level was also high. At P27, topo IIalpha was no longer detectable in both the DNA peak fractions and free protein fractions. On the other hand, topo IIbeta showed a triphasic time course during the postnatal development (Fig. 3B). In both its total amount and the DNA-linked forms, the enzyme gave a high signal already at P1 and after a transient decrease at P5, turned to increase again (second phase). The topo IIbeta -DNA complex stayed at a high level during the first 2 weeks, decreased gradually in the third week, and became almost undetectable at P27. Interestingly, the peak of granule cell differentiation in the second week coincides with the period when topo IIbeta is most active on chromatin. The total amount of topo IIbeta was also maintained at an elevated level during the first 2 weeks, then decreased to about half the maximum level at P20, and in contrast to topo IIalpha , this level was maintained at P27 (third phase) and even in adulthood. The time course of topo IIbeta expression was also confirmed by a conventional Western blotting of tissue lysates (Fig. 3B, open triangles).



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Fig. 3.   Time course of the topo II isoform expression and their in vivo activity during the postnatal cerebellar development. Cerebellar slices from P1 through P27 rats were incubated with or without 100 µM etoposide and the DNA-linked topo II was separated from free enzyme by CsCl density gradient centrifugation. Topo II isoforms in the gradient fractions were quantified as in Fig. 2. A, topo IIalpha ; B, topo IIbeta . Densitometric values plotted here had been normalized by total DNA amounts loaded on the gradient. Note the difference in the ordinate scale magnitudes. Topo II proteins cross-linked to DNA are shown by closed circles. Open circles indicate total topo II protein in the gradient. Open triangles in B designate relative amounts of topo IIbeta in tissue homogenates as determined directly by Western blotting.

To identify what cell populations contribute to the levels of total enzyme and their in vivo activities, spacial distribution of topo II isoforms in the developing cerebellar cortex was visualized by immunostaining (Fig. 4). The topo IIalpha -positive cells in EGL were already present at P1, increased in number to peak at P10, and decreased to disappear at P25 (Fig. 4A). This changing pattern closely parallels the monophasic time course of its in vivo activity (Fig. 3A), which can thus be ascribed to the granule cell progenitors proliferating in the mitotic zone of EGL. Topo IIalpha signal was not detected in Purkinje cells throughout the postnatal period, being consistent with the fact that their final cell division occurs in an embryonic stage. With respect to the in vivo activity of topo IIbeta (Fig. 3B), the initial phase is likely due to the multilayered Purkinje cells that constitute the major population of topo IIbeta -positive cells at P1 (Fig. 4A). The fluorescence and confocal microscopy at a higher magnification revealed that both nucleoplasm and nucleolus are brightly stained in P1 Purkinje cells (Fig. 4B). As they mature, the topo IIbeta signal decreased significantly in nucleoplasm but persisted in nucleoli. Granule cells should contribute little to the topo IIbeta activity at P1 since its population size is still small and granular layer is not generated yet. However, the second phase of the topo IIbeta activity that peaks around P9 is clearly accounted for by the growing population of differentiating granule cells. Granule cell nuclei in the granular layer were highly labeled with anti-topo IIbeta antibody at P10 but the signal was attenuated significantly at P25 when granule cells reach maturity (Fig. 4B). After the completion of cerebellar architecture, there is an intriguing discrepancy between the amount of topo IIbeta expressed in nuclei and that actually acting on DNA. At this stage, a large proportion of the enzyme appears to be localized in nucleoli (Fig. 4B).



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Fig. 4.   Immunolocalization of topo II isoforms during the postnatal cerebellar development. A, diaminobenzidine staining with the isoform-specific antibodies. Arrows indicate Purkinje cells. B, fluorescence micrographs of 4',6-diamidino-2-phenylindole (DAPI) staining and topo IIbeta staining (paired panels in the left) and confocal images for topo IIbeta staining (right panels). PL, Purkinje cell layer; ML, molecular layer; GL, granular layer; Pc, Purkinje cells; gc, granule cells.

Effects of Topo II-specific Inhibitors on the Granule Cells Differentiating in Vitro-- The topographical and chronological analyses on the levels of topo IIbeta expression and its in vivo activity suggested that the isoform beta  may play a critical role in the differentiation process of cerebellar neurons. To further investigate this possibility, we used a primary culture system for cerebellar granule cells that has been well established and used successfully in many studies. Expression of various genes required for neuronal maturation was shown to be tightly regulated in the course of culture, mimicking the in vivo situation (35, 36).

Granule cells prepared from P8 rat cerebellum by enzymatic dissociation extend processes in vitro to form elaborate neurite networks during the culture as shown by the staining with an antibody against neurofilaments (Fig. 5). When cells were stained with anti-topo IIalpha antibody, only a small proportion of cell nuclei (<4%) was positive for topo IIalpha after 1 day in culture (indicated by arrow). The positive cells decreased further in the following days. This is consistent with the notion that cells surviving in the culture are mostly postmitotic granule neurons. However, the level of topo IIbeta expression was already significant on day 1 in a large population of cell nuclei and appeared to increase as the cells differentiate in vitro, resembling the changing profile observed in vivo. The enhanced staining might be caused in part by an increased accessibility of the antibody since only a moderate increase in the levels of topo IIbeta protein and its mRNA was observed (data not shown).



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Fig. 5.   Expression of neurofilaments and topo II isoforms in the cerebellar granule cells differentiating in vitro. Cerebellar tissue from P8 rats was processed for primary culture according to the procedure described under "Experimental Procedures." Granule cells grown on coverslips were fixed at the days indicated with 4% paraformaldehyde and immunostained with antibodies to neurofilament proteins, topo IIalpha , and topo IIbeta .

Expression of representative genes underlying the differentiation phenotypes of the granule neuron were then analyzed. Amphiphysin I is a protein highly expressed in the central nervous system, including cerebellum, and involved in synaptic membrane recycling at nerve terminals (37). Levels of amphiphysin I was measured daily by immunoblotting (Fig. 6). The expression of amphiphysin I was markedly induced during the culture period, up to more than 10 times at day 5 as compared with day 1. However, in the continuous presence of ICRF-193, a topo II-specific inhibitor, the induction was suppressed significantly. Under these conditions topo IIbeta activity was inhibited to <5% of control.2 Similar effect on the amphiphysin I induction was exerted with etoposide at a noncytotoxic concentration. Although these inhibitors do not discriminate topo II isoforms, one can assume that topo IIbeta is the only target of the drug under these conditions in the absence of topo IIalpha expression. Immunocytochemical staining revealed that the induction of amphiphysin I in the cell body and neurite extensions was susceptible to ICRF-193 (not shown). It should be pointed out that those topo II inhibitors with different action mechanisms bring about similar results. ICRF-193 and other bisdioxopiperazines inhibit topo II activity without interfering with the DNA rejoining step but by binding to the closed-clamp form of the enzyme-ATP complex to prevent its conversion to the open-clamp form (38). Presence of cell lines that are selectively resistant to this class of drugs and express topo IIalpha having a point mutation in its ATP-binding site (39) strongly suggests that topo II is the single molecular target of ICRF-193. Also deserve mentioning here is that the cells stayed healthy and viable until the final day in culture in the presence of ICRF-193. Generally, it is much less cytotoxic compared with topo II poisons like etoposide which induce DNA strand breaks. Cytochemical staining of apoptotic cells detected only a low level of apoptosis in both the ICRF-treated and control cultures.



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Fig. 6.   Effects of topo II inhibitors on the induction of amphiphysin I in the granule cell culture. Cells were harvested at the times indicated and analyzed for protein expression by immunoblotting (control). In separate series of cultures, 1 µM etoposide (VP-16) or 10 µM ICRF-193 was added at the first, third, and fifth days to ensure continuous treatment. This treatment schedule was adopted to minimize the cytotoxicity of etoposide and maximize the effect of ICRF-193 which was shown to inhibit topo II activity in vitro completely at 10 µM (54) and inhibit the growth of U-937 cells for at least 3 days in culture with a single addition at 0.6 µM (55). Protein blots were probed with a human autoantibody against amphiphysin I. Immunoreactive bands on the membrane (shown below the graph) were scanned and quantified by densitometry. As upper bands of the doublet were identified as a phosphorylated form of amphiphysin I, they were also included into the calculation. Data points were normalized by the total protein amounts loaded onto the gel and expressed as induction folds relative to day 1.

In the experiment shown in Fig. 6, topo II inhibitors were first added at day 1 and cells were kept exposed to the drugs thereafter by supplementary additions. To see whether the inhibitory effect on amphiphysin I expression depends on the initiation timing of the drug treatment, the addition of ICRF-193 was delayed in order (Fig. 7A). The efficiency of inhibition decreased successively as the timing of drug addition was delayed. This phenomenon was more clearly illustrated by plotting the starting time of drug treatment against a susceptibility index which was calculated from the induction rate determined after 1.5 days (Fig. 7B). These data suggest that topo IIbeta activity is involved in an early stage of granule cell differentiation in vitro that potentiates the induction process of amphiphysin I. Topo IIbeta activity appears to be essential for this stage to initiate since even in cells cultured for 4.5 days in the presence of ICRF-193, amphiphysin I was induced at a normal rate upon removal of the drug (Fig. 7A, f).



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Fig. 7.   Effects of delayed addition or removal of ICRF-193 on the amphiphysin I induction. A, amphiphysin levels were analyzed as described in the legend to Fig. 6. No drug control (a); 10 µM ICRF-193 was added daily starting from the culture days 0.5 (b), 2.5 (c), 4.5 (d), and 6.5 (e). In f, cells were treated with the drug as in b until day 4.5 when the medium was replaced with drug-free medium. B, ICRF susceptibility of the amphiphysin induction was calculated by {1 - (increment during 1.5 days in culture with drug)/(increment during 1.5 days in culture without drug)} × 100, and plotted against the starting time of the drug treatment. One-hundred % susceptibility indicates complete repression of the amphiphysin induction during the first 1.5 days in the presence of ICRF-193.

The induction of amphiphysin I protein was reflected by the mRNA contents as estimated by Northern blotting (Fig. 8A). The amphiphysin I transcript was also decreased by ICRF-193 to a similar extent as in the protein level, suggesting that the effects of topo II inhibitors on protein amounts are not due to a decreased rate of translation or an accelerated protein degradation. Effects of ICRF-193 on transcript levels were further analyzed with other gene probes and are also shown in Fig. 8. All the transcripts were induced as granule cells differentiate in culture. These proteins are directly involved in neuronal functions such as exocytosis (synaptophysin) or endocytosis (amphiphysins I and II) of synaptic vesicles, and a neurotransmitter receptor protein (gamma -aminobutyric acid receptor alpha 6 subunit). The inducible genes were either susceptible to the drug (Fig. 8, A and B) or not susceptible at all (Fig. 8, C and D). Genes that are already expressed in vivo at P8 and some housekeeping genes tested so far were not suppressed by ICRF-193 (data not shown). Amphiphysin II is an isoform of amphiphysin I encoded by a distinct gene (37). They have been suggested to cooperate by forming a heterodimer (40). Unexpectedly, they behave differently in the presence of the topo II inhibitor (Fig. 8, A and C). These results from the transcript analysis were confirmed at the protein level by Western blotting.



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Fig. 8.   Differential effects of ICRF-193 on the transcript levels of neuronal genes that are induced in granule cells. Total cellular RNA extracted from control cells or the cells treated with 10 µM ICRF-193 as described in the legend to Fig. 6 was subjected to Northern hybridization with 32P-labeled cDNA probes: A, amphiphysin I; B, synaptophysin; C, amphiphysin II; and D, gamma -aminobutyric acid receptor alpha 6. Radioactive mRNA bands visualized on the autoradiogram are shown in the inset (-, control; +, ICRF-treated). Bands were scanned, quantified by densitometry, and the relative densitometric units, day 1 as unity, were plotted (open circles, control; closed circles, ICRF-treated).

If the mRNAs for amphiphysin I and synaptophysin are rapidly turning over, the observed susceptibility to ICRF-193 might be caused by a slight decrease in their transcription rates. To see whether or not this is the case, mRNA turnover rates were measured by blocking the RNA polymerase II-dependent transcription with 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole at an early stage of chain elongation (Fig. 9). Half-lives were estimated to be 10 and 14.3 h for amphiphysin I and synaptophysin mRNAs, respectively, indicating that both messages are quite stable. Also, ICRF-193 did not facilitate their degradation since the turnover rates remained basically unchanged in its presence. Furthermore, the drug exerted little effect on the transcription initiation of both genes in late stage granule cells, as revealed by a nuclear run-on assay in that the cells at culture day 6 were treated with ICRF-193 for 24 h (not shown). This is also consistent with the results at the protein level (Fig. 7). These data suggest that the differential effect of ICRF-193 on mRNA levels is not due to differences in mRNA turnover rates. Therefore, it is most conceivable that the inhibition of topo IIbeta activity interferes with some upstream events, resulting in the selective suppression on the transcriptional induction of a subset of inducible genes.



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Fig. 9.   Determination of mRNA turnover rates in the presence and absence of ICRF-193. Granule cells at day 5 in culture were treated with 20 µg/ml 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB) to arrest general mRNA synthesis. To one-half of the culture plates, 10 µM ICRF-193 was also added. Total cellular RNA was prepared after 0, 3, 6, 12, and 24 h of incubation, and then subjected to Northern analysis as in Fig. 8 with labeled probes for amphiphysin I (A) or synaptophysin (B). From the regression lines to semi-logarithmic plots (without ICRF treatment), half-lives of mRNA for amphiphysin I and synaptophysin were calculated to be 10.0 and 14.3 h, respectively.

As only a limited number of gene transcripts were analyzed thus far, we finally investigated inducible genes and their ICRF susceptibility using a larger set of identified gene probes that are fixed on membrane as a cDNA macroarray. Out of 300 detectable hybridization signals, 54 (18%) were increased, 36 (12%) were decreased, and 210 (70%) were unchanged during the 5 days in culture. The identity of up-regulated genes were summarized in decreasing order of inducibility (Fig. 10). As expected, multiple genes that are required for neuronal functions such as ion channels, receptors, and signal transduction molecules were noticeable in the list. Although ICRF-193 showed little effect on majority (96%) of the constitutively expressed genes, one-third of the inducible gene transcripts were repressed by the drug (termed class I genes). In light of these results, we propose that inducible genes can be classified into two groups with respect to their susceptibility to ICRF-193 in vitro. According to this criteria, therefore, amphiphysin I and synaptophysin belong to the class I, and amphiphysin II and gamma -aminobutyric acid receptor alpha 6 belong to the class II genes (Fig. 8). The underlying mechanism for the difference is unclear at present (see "Discussion"). In conclusion, topo IIbeta may thus be involved in an early stage of differentiation and regulate the transcriptional induction of a subset of neuronal genes that are to be up-regulated during the course of differentiation.



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Fig. 10.   Identification and ICRF susceptibility of the genes that are up-regulated during the granule cell differentiation in vitro. Three RNA preparations were isolated from untreated granule cells at day 1, at day 5, and ICRF-treated cells at day 5, respectively. Relative levels of specific transcripts in these preparations were determined by hybridization to a cDNA macroarray. Gene transcripts that displayed 2-fold or greater changes were scored as significant changes. Only 36 induced genes are shown here with GenBankTM accession number, protein identity, induction fold, and the ICRF-inhibition index defined as {(signal increment during 5 days in culture with drug)/(signal increment during 5 days in culture without drug)} × 100. The inducible genes were sorted into two groups with different susceptibilities to ICRF-193 by applying preset thresholds for the ICRF-inhibition index: less than 50% (class I genes), and between 70 and 130% (class II genes).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Topo II Isoforms in Differentiating Neuronal Cells Are Catalytically Active in Vivo-- Topo II is an enzyme that transiently cleaves DNA double strands in one duplex segment to form an enzyme-operated gate and rejoins the cleaved DNA ends after passing a second double-stranded DNA segment through the gate. This enzymatic activity is essential for the segregation of daughter chromosomes and thus for cell proliferation. Growing evidence supports that the role is exclusively played by topo IIalpha in mammalian cells (1). Our immunohistochemical data showing that topo IIalpha in the developing cerebellum localizes predominantly in cells situated in the mitotic zone are consistent with this view. A large proportion of topo IIalpha was found in covalent complexes with DNA after the treatment with etoposide, suggesting that topo IIalpha expressed in dividing EGL cells is catalytically involved in vivo in proliferative processes such as chromosomal condensation and segregation (41, 42).

Following the final division cycle and migration in embryonic days, Purkinje cells start to develop dendrites at their final destination beneath the EGL (34). It is these periods (P1-P5) when the expression of topo IIbeta in nucleoplasm of Purkinje cells reaches the highest level. Immunofluorescence and confocal microscopy revealed fine granulo-fibrous or reticulo-punctate localization of topo IIbeta throughout the nucleoplasm. Similar nucleoplasmic distribution of topo IIbeta was reported in cultured cells such as A431 human epidermoid cells (43) and Chinese hamster ovary fibroblasts (44). The transient decrease in the total amount of topo IIbeta that occurred at around P5 (Fig. 3) may be caused by the declining topo IIbeta content in Purkinje cells. At P5, topo IIbeta expressed in granule cells seems to contribute only partially to the total enzyme amount.

Granule cells start to differentiate in the inner zone of EGL (differentiating zone) soon after their final division in the mitotic zone of EGL. The granule cell differentiation is most active around 10 days after birth (34). The expression of topo IIbeta in the nucleoplasm follows a similar time course and so does the immunoreactive material recovered from the CsCl gradient (Fig. 3). Intranuclear localization of topo IIbeta showed a fine granulo-fibrous pattern as seen in the differentiating Purkinje cells. In both Purkinje cells and granule cells, therefore, intensity of topo IIbeta staining in the nucleoplasm increased as cellular differentiation proceeds, reaching a maximal level when the cells are most actively involved in differentiation. At the peak of granule cell differentiation, about a quarter of total topo IIbeta was recovered as topo II-DNA covalent complexes after treating the cerebellar tissue with etoposide. This is a clear indication of topo IIbeta being actively engaged in its catalytic reaction in the differentiating neuronal cells. Since the gene expression in developing cerebellum is strictly controlled in a temporo-spatial manner (45), one can speculate that topo IIbeta in differentiating neurons might be a part of machinery that controls gene expression according to the developmental program, probably through altering the superstructure of genomic DNA. As the cerebellar architecture came close to its fully developed adult form toward the end of the third week, the catalytically competent topo IIbeta decreased gradually to reach a detection limit and the nucleoplasmic topo IIbeta in granule cells also decreased while the staining of nucleolus-like structures remained unchanged. Their identity as nucleoli has been demonstrated by double immunostaining of antigens that are known to localize in nucleolus.3 Expressed level of topo IIbeta protein also decreased in the third postnatal week, but the decrease leveled off at about half the maximum level. These results imply that the topo IIbeta in nucleoplasm is the catalytically competent entity that is actively involved in the neuronal differentiation and disappears once neurons are terminally differentiated. The residual topo IIbeta in the terminally differentiated neurons that localizes mainly in nucleoli is likely to be inaccessible to chromatin DNA although it is still active on naked DNA in vitro (16).

Functional Topo IIbeta May be Required for the Induction of a Subset of Neuronal Genes-- Topo II-specific inhibitors have been used in various cell systems to investigate the involvement of topo II in cell differentiation. However, the results from these studies, especially those done in early times without knowing the presence of isoforms, are difficult to interpret. Additional ambiguity originates from the fact that usually both topo II isoforms are expressed in cultured cells and they are both susceptible to such inhibitors. This makes it unable to assess which enzyme is responsible for modulated gene expression although levels of topo IIalpha and topo IIbeta can be estimated separately using isoform-specific antibodies. For instance, etoposide was shown to induce the differentiation of human promonocytic leukemia U-937 cells as revealed by an increase in the production of reactive oxygen species and surface expression of differentiation-specific antigens (46). It was also shown that the cells treated with etoposide were arrested at the G2 phase and ceased to proliferate. It appears, therefore, that the observed alteration in gene expression is attributable to the growth arrest caused by inhibition of topo IIalpha and similar effects may be expected when etoposide is replaced with mitotic inhibitors such as mitomycin C (47). Topo IIbeta may have some action points in differentiation pathways downstream the growth arrest but it is difficult to prove this because of the inherent experimental drawback mentioned above. The same situation applies to other studies using topo II inhibitors in differentiating culture cells (48, 49).

Our experimental system, primary culture of cerebellar granule cells, reflects an ongoing process of in vivo differentiation and is clearly advantageous over cultured cell lines that are forced to differentiate by addition of inducers. In the granule cell culture majority of cells that can be maintained in vitro are derived from the postmitotic neurons in EGL and possibly in ML but the cells in GL do not survive at all (50). Because of this selection, the culture starts with largely immature granule cells and their differentiation proceeds semi-synchronously. The present study demonstrates that the transcriptional induction of a number of genes is markedly repressed by topo II inhibitors (termed class I genes). The effect is caused neither by inhibition of topo IIalpha which is not expressed in these cells nor by inhibition of general transcription as supported by the predominance of class II genes and many other genes that are constitutively expressed and not susceptible to ICRF-193. The incomplete inhibition of class I genes may be explained by a subtle difference in the timing between the onset of induction and the addition of topo II inhibitors. As substantial topo IIbeta expression is already present in many cells at day 1 (Fig. 5), the putative process requiring topo IIbeta activity might have been partially completed when inhibitors were added, being consistent with the result that ICRF-193 exerted less effect on the amphiphysin I induction when the addition was further delayed (Fig. 7). The result also implies that the action point of the drug is not in the transcription per se which is still active in the later stages of differentiation and should be equally susceptible to the drug. These observations are consistent with the involvement of topo IIbeta in early processes of granule cell differentiation when class I genes are likely to be potentiated for transcription. The potentiation step for class II genes is either completed already in vivo or not required at all for their transcriptional activation. To our knowledge these results are the first unequivocal implication that catalytically competent topo IIbeta is required for a transcriptional regulation of differentiation-related genes. Attempts to clarify the molecular mechanisms underlying these processes are in progress.

Our study also corroborates a probable link between topo IIbeta and embryonic neural development in light of the recent report on a gene disruption mutant for topo IIbeta (24). The top2beta -/- mouse embryos show no apparent defect in neurogenesis and overall growth until E15.5. At E18.5, however, normal innervation pattern of the diaphragm and limb muscles is absent in that motor neuron axons fail to branch and contact muscle fibers. Development of sensory axons in the spinal cord is also defective in the top2beta -/- embryos. It is interesting to note that the mutation affects only late stages of embryogenesis and not all the neural developments are involved since the growth of motor axons is not impaired in the intercostal muscles. Therefore, topo IIbeta is not a general but rather a specialized factor that is essential for neuronal differentiation. This view is consistent with our results that induction of only a subset of genes is interfered by topo II inhibitors in the granule cell culture. The mutant mice die shortly after birth and it is not feasible to analyze the postnatal events including cerebellar development. Unfortunately, abnormalities in the central nervous system of the top2beta -/- embryos have not been assessed in detail. The absence of topo IIbeta may as well affect the early development of the Purkinje cell lineage occurring around E15, as we have observed a concomitant induction of topo IIbeta and calbindin, a marker protein for Purkinje cells, in the postmitotic progenitor cells.3

Possible Mechanisms for the Topo IIbeta -mediated Gene Regulation-- We have shown in the present study that the nucleoplasmic topo IIbeta is actively involved in DNA transactions underlying the granule cell differentiation that accompany activations of gene expression. Induction of developmentally regulated genes like the beta -globin gene cluster is believed to proceed in two steps (51). Prior to the transcriptional activation enabled by specific interactions between regulatory cis elements and trans-acting protein factors, chromatin conformation of the gene-containing region must be modified to a less condensed state to ensure such interactions (gene potentiation). Although there have been no direct evidence for chromatin decondensation catalyzed by topo IIbeta , the decondensation of sperm head chromatin in Xenopus egg interphase extract was shown to be inhibited by ICRF-193 (52). It is quite possible that topo IIbeta assists the decondensation of chromatin which is essential for some genes to be expressed according to the developmental program.

In eukaryotes, particular genomic segments called matrix attachment regions organize the genome into large looped domains that are topologically independent. We have demonstrated recently that the putative action sites of topo IIbeta in the P10 rat cerebellum are predominantly AT-rich noncoding sequences frequently associated with matrix attachment regions.4 Topological effects exerted by topo IIbeta acting on matrix attachment regions can be propagated throughout the whole loop. We also found that the average nuclear volume of granule cells increases by 30% after 7 days in culture (results not shown). Nuclear size of nondividing cells is generally correlated with the state of chromatin condensation and the differentiating granule cells indeed showed more dispersed chromatin when DNA was stained with Hoechst 33342. Decondensation of chromatin have also been noticed in the maturation process of granule cells observed in tissue sections (34). As expected, the nuclear enlargement in vitro was suppressed by ICRF-193. Therefore, topo IIbeta appears to modulate the superstructure of chromatin in a detectable scale. This may involve the nucleosomal structure as well, because a recent study showed that Drosophila topo II is a component of a chromatin-remodeling factor designated CHRAC (53). Connection between the chromatin decondensation and the transcriptional induction of specified genes will be demonstrated by further analysis including the detection of altered chromatin conformation and detailed mapping of topo IIbeta target sites within the class I gene. These experiments will reveal the local environment of individual genes and also clarify the reason why the induction of class II genes is independent of topo IIbeta activity.


    ACKNOWLEDGEMENTS

We thank N. Nozaki (Kanagawa Dental College) for providing the topo II isoform-specific monoclonal antibodies and M. Miyaike (Mitsubishi Kasei Institute of Life Sciences) for determining the epitope regions of these antibodies. We are grateful to R. Takeuchi and M. Ishimaru for technical assistance.


    FOOTNOTES

* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (project number 11239206).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel.: 81-86-235-7386; Fax: 81-86-235-7392; E-mail: tsukken@cc.okayama-u.ac.jp.

Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008517200

2 K. Sano, K. Tsutsui, and K. Tsutsui, unpublished observation.

3 K. Tsutsui, K. Tsutsui, O. Hosoya, K. Sano, and A. Tokunaga, J. Comp. Neurol. (2001), in press.

4 K. Tsutsui, K. Sano, K. Tsutsui, A. Kikuchi, and A. Tokunaga, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: topo II, DNA topoisomerase II; EGL, external germinal layer; PBS, phosphate-buffered saline.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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