DNA topoisomerase I (Topo I) specifically phosphorylates arginine-serine-rich (SR proteins) splicing factors and is potentially involved in pre-mRNA-splicing regulation. Using a Topo I-deficient murine B lymphoma-derived subclone (P388-45/C) selected for its resistance to high dosage of the antitumor drug camptothecin, we show that Topo I depletion results in the hypophosphorylation of SR proteins and impairs exonic splicing enhancer (ESE)-dependent but not constitutive splicing. The Affymetrix GeneChip system analysis revealed that several alternatively spliced genes, characterized by small exons and large introns, are down-regulated in Topo I-deficient cells. Given that ectopic expression of green fluorescent protein-Topo I fusion in Topo I-deficient cells restores both wild-type phosphorylation of SR proteins and ESE-dependent splicing, we conclude that Topo I-mediated phosphorylation plays a specific role in ESE-regulated splicing.

Human Topo I4 plays a key role in many fundamental biological processes such as transcription and DNA replication and repair (1). This enzyme alters DNA topology by transiently breaking and resealing one strand of DNA through a covalent protein-DNA intermediate (2), and both positive and negative supercoils can be relaxed by human Topo I. It also demonstrates an intrinsic protein kinase activity (Topo I kinase; Refs. 3, 4) required to achieve specific phosphorylation of SR protein splicing factors in vitro. Accordingly, human DNA Topo I has been shown to exhibit at least two incompatible conformations. One conformation is in the form of a complex with one of the substrates of the kinase reaction (ATP or the SR splicing factor ASF/SF2), which inhibits DNA relaxation activity, and the other, a Topo I-DNA complex, which inhibits protein kinase activity (5, 6). The SR proteins are part of a growing family of structurally related and highly conserved splicing factors characterized by the presence of one or two RNA recognition motifs and a COOH-terminal arginine-serine repeat of varying length (RS domain; Refs. 7, 8). The RNA recognition motifs mediate recognition of specific RNA motifs known as ESEs, which play a key role in both alternative and constitutive splice site selection. Therefore, Topo I could potentially be involved in controlling the splicing of mRNA precursors (pre-mRNAs), in addition to its role in transcription and replication. Along this line, both hyper- and hypophosphorylation of SR proteins were shown to inhibit splicing (see Ref. 9 for review), and specific inhibitors of the Topo I kinase activity (10) led to altered splicing pattern of several genes, among which Bcl-X and CD 44 known to affect apoptosis and tumor progression (11). However, the exact role of Topo I-mediated phosphorylation on splicing regulation is not known, and how Topo I exerts its dual function in vivo is not well understood.

DNA Topo I is also an important target for cancer chemotherapy. Levels of this enzyme are elevated in several types of leukemia, lymphoma, and colon carcinoma cells (12), and a variety of promising antineoplastic agents that inhibit Topo I activity are currently under examination (13, 14). Several such agents are either derived from the cytotoxic plant alkaloid CPT or from indolocarbazole derivatives (15). CPT is a nonintercalating inhibitor that primarily acts by inhibiting the rate of DNA religation and, therefore, by reversibly stabilizing a covalent intermediate between the 3′ phosphate end of the cleaved DNA strand and tyrosine 723 at the active site (16, 17). The formation of this Topo-drug-DNA ternary complex, called cleavable complex, appears to be critical for the cytotoxic effect of CPT (13, 18). Stepwise exposure of several cell types to increasing levels of CPT has resulted in the isolation of drug-resistant cell lines exhibiting reduced Topo I levels, mutations in the coding region of the gene, or both (4, 13).

In this study, we have taken advantage of a cellular clone (P388-45/C) derived from the P388/CPT45 cell line (19, 20) and which shows hardly any detectable DNA Topo I to investigate the function of this enzyme in splicing. We show that Topo I-deficient cells accumulate hypophosphorylated variants of SR proteins and are defective in ESE-dependent but not constitutive splicing. Ectopic expression of a GFP-Topo I fusion protein restores both wild-type levels of SR protein phosphorylation and the ability of P388-45/C cells to perform ESE-dependent splicing. These results demonstrate that the kinase activity of Topo I plays an important role in the regulation of ESE-dependent splicing.

Cell Culture Conditions and Transfection Procedures.

The P388 murine leukemia cell line was obtained from the tumor bank of the National Cancer Institute (Bethesda, MD). The P388-45/C subclone was derived from the P388/CPT45 cell line (19, 20) after two rounds of subcloning selection in the presence of increasing CPT concentrations as follows: P388-45R cells [resistant to 45 μm CPT; a gift from Michael R. Mattern, Smith Kline Beecham (Kings of Prussia, PA)] grown in RPMI 1640 containing 0.01 mm 2-mercaptoethanol, 10 mml-glutamine, 100 IU/ml penicillin, 2 μg/ml streptomycin, 50 μg/ml gentamicin, 50 μg/ml nystatin, 30% fetal bovine serum, and 10 μm CPT were seeded in 96-well plates and incubated at 37°C in a humidified atmosphere containing 5% CO2. Colonies were grown for 3 months with continuous CPT exposure, checked several times for depletion of Topo I band by Western blot. Few clones that show hardly any detectable Topo I band by Western blot with mAbs were seeded into new 96-well plates and continuously treated with either 10 or 45 μm CPT. Few clones were selected from 10 μm CPT-treated cells and one clone from 45 μm CPT-treated cells. The latter clone named P388-45R/C was grown for 4 months with continuous 45 μm CPT treatment. At this stage, the P388-45R/C clone was grown without CPT for 10 passages, and the Topo I protein did not return. The doubling times of P388 and P388-45/C cells were 13 and 22 h, respectively.

Transfections were performed with the DMRIE-C transfection reagent (Invitrogen) essentially as recommended by the supplier. Briefly, 5 × 106 cells were transfected with 10 μg of plasmidic DNA and 15 μl of DMRIE-C in serum free OPTI-MEM medium. After a 5-h incubation at 37°C, complete RPMI medium was added to the transfected cells. For stable transfection experiments with the GFP-hTop expression vector, G418 (600 μg/ml final concentration; Invitrogen) was added to the cells 72 h after transfection, and selection pressure was maintained thereafter.

Topo Assays.

The DNA relaxation activity present in various amounts (3–15 μg) of NE purified from P388 and P388-45/C cells was analyzed as described previously (21).

Western Blot and Two-Dimensional Gel Analyses.

NEs samples in Laemmli loading buffer were run on 10% SDS-polyacrylamide gel and transferred onto nitrocellulose by electroblotting for 90 min in 10 mm 3-(cyclohexylamino)propanesulfonic acid (pH 11.0) transfer buffer containing 10% methanol. Blots were probed either with monoclonal anti-GFP (Roche Diagnostics) monoclonal or polyclonal anti-Topo I antibodies and revealed by enhanced chemiluminescence.

NEs (600 μg) purified from P388, P388-45/C, 45/C-GFP, and 45/C-GFP Topo cells were subjected to two-dimensional analysis with Immobiline Drystrip (180 mm, pH 3-10L) using a Multiphor II electrophoresis unit (Amersham Pharmacia Biotech) according to manufacturer’s instructions. Isoelectric focusing (500 V for 1 h, 5000 V for 8 h, and 300 V for 10 h) was performed at 20°C after a 12-h rehydration step. After the second dimension electrophoresis, proteins were transferred onto nitrocellulose, and blots were probed with the indicated mAbs.

NEs Preparation, Splicing, and Complementation Assays.

NEs were prepared according to Ref. 22. The Minx (23), pSpHbm3S1, and DUP51 3S (24) have been described previously. Pre-mRNA was synthesized by in vitro transcription in the presence of 20 units of SP6 (Boehringer) or T7 (Invitrogen) RNA polymerase, 1 μg of the suitable linearized plasmids, and 5 μm [α-32P]UTP (3000 Ci/mmol) in 25-μl reactions according to manufacturer conditions. In vitro transcripts were quantified by Cerenkov counting. Splicing reactions were performed under standard conditions as described previously (25). Splicing products were analyzed by electrophoresis on denaturing polyacrylamide gels and revealed by autoradiography.

ASF/SF2 proteins used in complementation assays were produced and purified from baculovirus-infected Sf9 cells as described previously (3). Differentially phosphorylated ASF/SF2 proteins were obtained in fractions corresponding to different steps of the elution procedure. The Hexahistidine-tagged ASF/SF2 protein was also produced and purified from Escherichia coli as described previously (26).

Determination of Expression Levels Using RT-PCR Assay.

Total RNA was purified from P388, P388-45/C, and 45/C-GFP Topo cells by the Tri-Reagent (Sigma) procedure. First strand cDNA was synthesized from 5 μg of DNase-treated RNA with the Amersham-Pharmacia cDNA synthesis kit. For PCR analyses, one-tenth of the reaction was amplified with Taq polymerase (Invitrogen), and the cycle number was kept to a minimum to maintain linearity. PCR products were separated on 1.5 or 1.0% agarose gels containing ethidium bromide and visualized under UV light. PCR regimes and nucleotidic sequences of the primers used in this study are available upon request.

Affymetrix GeneChip Expression Analysis.

For analysis of gene expression, two independent preparations of total RNA obtained from P388 and P388-45/C cells by the Tri-Reagent procedure were used. After isolation of total RNA, double-stranded cDNA was synthesized by reverse transcriptase (Life Technologies, Inc.) using a T7-(dT)24 primer. Biotin-labeled antisense cRNA was transcribed from cDNA templates using T7 RNA polymerase (Enzo Biochem) and hybridized to oligonucleotide microarrays (MG_U74Av2 murine array; Affymetrix), each containing 12,488 functional oligonucleotide probe sets. Typically, 16 pairs of 25-mer oligonucleotides are used to interrogate the transcript level of the genes represented on the array. Each probe pair consists of a PM complementary to the target sequence and a mismatch oligonucleotide, which differs from its PM counterpart by a single base in the center of the sequence. The probe array and the hybridization levels were measured with the Agilent GeneArray scanner after staining of the array with streptavidin-phycoerythrin. Hybridization intensities (average differences) and assessments of presence or absence of a given transcript (absolute call) were determined with Affymetrix Microarray Suite 4.0 (Affymetrix, Inc.) using Affymetrix default parameters. Expression algorithms computes two main absolute metrics, an absolute call (absent, present, or marginal) for the significance of gene presence and a gene expression level (average difference) calculated as the average of the PM-mismatch oligonucleotide differences for all probe pairs in a probe set. Comparative expression results are given as a difference call (increase, decrease, or no change) for the significance of expression differences and a fold change indicating the relative change in the expression levels. Resulting Affymetrix text files were compared with Genespring software by using the following criteria: maximum hybridization intensity > 200 in at least one of the comparison samples; a fold change between normalized data from compared samples of at least three; and statistical significance among replicates. Clustering of genes into different categories was done manually based on published available data.

Characterization of CPT-Resistant Cell Line Deficient in Topo I.

The P388-45/C CPT-resistant cell line was obtained by two subcloning steps of the P388-CPT45 derivative of P388 mouse leukemia cells (19, 20) to stepwise increasing concentrations of CPT until they grew in the presence of 45 μm CPT, a concentration 450 times higher than normally used to kill parental cells. The ability of a P388-45/C cell to overcome the myriad of deleterious side effects responsible for CPT cytotoxicity depends on mechanisms reducing cleavable complex formation. Among such mechanisms are those leading to a reduction of DNA Topo I protein expression. Whether DNA Topo I expression is altered in P388-45/C cells was first tackled by comparing the DNA relaxation activity in NEs prepared from parental and resistant cells. As shown in Fig. 1,A, the supercoiling of the plasmid DNA used in this assay was not significantly affected upon incubation in P388-45/C NE, whereas it was almost completely relaxed in NE from parental cells. That this lack of activity results from a dramatic down-regulation of DNA Topo I expression in P388-45/C-resistant cells was established by Western blot analysis of Topo I levels in P388 and P388-45/C cells (Fig. 1,B). Neither monoclonal (Fig. 1,B, left panel) nor polyclonal (Fig. 1,B, right panel) antibodies raised against Topo I were able to detect any traces of this protein in P388-45/C NE (line 2), demonstrating that depletion of Topo I is at the origin of resistance of P388-45/C cells to high dosage of CPT. Because RT-PCR (data not shown), as well as Affymetrix GenChip analysis (Table 1), indicate that Topo I mRNA level is also significantly reduced but not completely abolished in P388-45/C, it is likely that posttranslational mechanism(s) be also responsible for the lack of Topo I protein expression. Given that P388-45/C only have an increased doubling time (40 h compared with 16 h for P388), these data establish that, as with yeast, mammalian Topo I is not required for viability.

Transcriptional Activity of Topo I-Deficient Cells.

Because of its ability to remove positive and negative supercoils from constrained DNA, Topo I has been proposed to provide swivels for removing torsional constrain that accompany the transcription process (1, 2, 27). To test the requirement for Topo I in transcription, we performed a differential analysis of mRNAs isolated from P388 and P388-45/C cells. Biotinylated cRNAs were hybridized to the Affymetrix Mouse Gene Chip Mu74 containing >12,000 oligonucleotide probe sets and, after elimination of data points that were suspected because of low signal or high background, hybridization signals generated from P388 and P388-45/C RNA sets were compared. This study revealed that only few genes (∼216 genes) were either up-regulated or down-regulated in cells lacking Topo I. Interestingly, the same set of genes was found to follow the same regulation in another CPT-resistant clone (data not shown), which also demonstrated low levels of Topo I, indicating that expression of these transcripts is influenced by the levels of Topo I and/or CPT treatment. Among the expressed sequence tags that were either up-regulated or down-regulated, 32 and 45%, respectively, were of unknown function. Noteworthy, the number of genes in which the transcript level underwent at least 3-fold increase relative to those from P388 cells in the two experiments is very close to that of down-regulated genes (Tables 1 and 2 and data not shown), indicating therefore that lack of Topo I does not affect transcription in a uniform way. Microarray results were confirmed by semiquantitative RT-PCR analysis of transcripts sampled from various categories. These include examples of up-regulated (Casp 6, interleukin 1 receptor, CD 2 and Ly 6, Fig. 2,A) and down-regulated mRNAs (lamin A, histone H1, β-globin, and CD24; Fig. 2 B). Affymetrix data also reveal that genes whose expression is either up-regulated or down-regulated do not belong to a restricted subset of functional classes but rather correspond to functionally related classes involved in immune response, cell proliferation, differentiation and signaling, or coding for transcription factors, DNA binding proteins, enzymes, and structural proteins.

Although all genes showing either increase or decrease in P388-45/C cells bear no obvious connection to Topo I function in transcription regulation, the robust up-regulation of cytokines and their receptors, together with the down-regulation of cyclin E, cyclin-dependent kinase 5, protein kinase C, and Topo I genes, strongly argue that P388-45/C cells have accumulated survival signals allowing them to overcome deleterious effects of CPT (see “Discussion”).

Topo I-Deficient Cells Contain Hypophosphorylated SR Proteins.

Intriguingly, a BLAST-Like Alignment Tool (BLAT) search revealed that several down-regulated genes in Topo I-deficient cells are characterized by small exons and large introns (see “Discussion”) and can undergo alternative splicing (Table 1), consistent with the hypothesis that Topo I might be involved in pre-mRNA splicing. Because Topo I phosphorylates SR proteins in vivo, it can be expected that its absence affects the overall phosphorylation level of SR proteins and, thereby, their splicing activity. To test this possibility, NE prepared from P388 and P388-45/C cells were resolved on two-dimensional gels, and SR protein phosphorylation variants were analyzed by Western blot using first a mAb (mAb 104), which recognizes a phosphorylated epitope at arginine/serine rich (RS) domain. Although mAb 104 is expected to reveal several SR protein species (28) defined as SRp20, SRp30a (ASF/SF2), SRp30b (SC35), 9G8, SRp30c, SRp40 [hepatic arginine/serine protein (HRS)], SRp55, and SRp75 depending on their apparent molecular weight in SDS gels, strong bands were detected at the level of SRp30 but weak signals at the level of SRp40, SRp55, and SRp75 in P388 (Fig. 3,A, left panel). This phenomenon is exacerbated in P388-45/C, where no signal was detected at level of SRp40, SRp55, or SRp75 (Fig. 3,A, right panel). Even the number of SRp30 phosphorylated variants appears to be reduced compared with those from P388, suggesting that in Topo I-deficient cells, some SR protein phosphorylation isoforms might have lost the phosphoepitope recognized by mAb104 (Fig. 3,A, compare left and right panels). To test this more directly, we then used specific antibodies raised against ASF/SF2, SC35, and 9G8, which contribute to the SRp30 signal. Whereas the 9G8-specific antibody failed to detected any signal in P388 and P388-45/C, implying that this protein is absent from these cells (data not shown), five ASF/SF2 phosphorylation variants were detected in P388 (Fig. 3,B, top panel) and six in P388-45/C (Fig. 3,B, bottom panel). Careful comparison of the mobility of each variant relative to internal abundant proteins demonstrated that the most phosphorylated variant of ASF/SF2 (referred to as position 0) in P388 is absent in P388-45/C (Fig. 3,B, compare top and bottom panels). In contrast, although less phosphorylated variants at positions −5, −6, and to a lesser extent −7 are abundant in P388-45/C, they are absent in P388 (Fig. 3,B, compare top and bottom panels). Similar results were obtained with SC35 antibody (Fig. 3,C). In this case, phosphorylation variants from P388 cells form a large smear compared with those from P388-45/C (Fig. 3,C, compare top and bottom panels) and the smear corresponding to most phosphorylated variants is absent from the P388-45/C pattern (Fig. 3 C, bottom panel). Thus, Topo I-deficient cells accumulate hypophosphorylated forms of ASF/SF2 and SC35. This result is in perfect agreement with a previous study showing that inhibitors of Topo I prevent hyperphosphorylation of SR proteins (3).

Splicing Activity of NEs from Topo I-Deficient Cells.

Phosphorylation status of ASF/SF2 has previously been shown to affect several properties of this protein (29, 30). The finding that SR proteins are differentially phosphorylated in P388 and P388-45/C cells prompted us to compare the in vitro splicing activity of extracts from both cell lines. For this study, we used synthetic mRNA precursors derived from either the adenovirus major late-transcription unit (Minx, Fig. 4,A) or the Drosophila fushi tarazu (ftz) gene (data not shown), which are single intron pre-mRNAs not requiring ESE sequences in the second exon for efficient splicing. As shown in Fig. 4 A, Minx pre-mRNA splicing efficiency is quite similar in both P388 (Lanes 1 and 2) and P388-45/C (Lanes 3 and 4) NE. The same results were obtained with the ftz-derived transcript, implying that SR proteins lacking Topo I-dependent phosphophorylation function in the basic splicing reactions.

In sharp contrast, P388-45/C NE failed to support efficient splicing of pre-mRNA substrates, the splicing of which depends on an ESE in the second exon. Neither the βglo 3S (pSpHβm-3S1) that has three copies of a high-affinity binding site for ASF/SF2 established by SELEX analysis (Ref. 24; Fig. 4,B, Lanes 3 and 4), nor E1A derivative-specific pre-mRNAs (Sp1-ASF; Ref. 31), in which the second exon contains a single copy of a high-affinity target for ASF/SF2 (data not shown), were efficiently spliced in extracts derived from P388-45/C, while they demonstrated efficient splicing in P388 NE (Fig. 4 B, Lanes 1 and 2 and data not shown).

ASF/SF2 Phosphorylation Levels and ESE-Dependent Inclusion of Alternative Exons.

Whether Topo I-mediated phosphorylation might be required for the regulation of alternative splicing was assessed by using a model pre-mRNA containing two introns and three exons derived from the human β-globin gene. This substrate (DUP51 3S referred here as DUP 3S) has been successfully used to demonstrate ESE-dependent inclusion of a small internal exon (24). Strikingly, although in P388 NE DUP 3S is spliced by a pathway that generates a fully spliced product containing all three exons (Fig. 5,A, Lanes 1 and 2), only a very weak signal corresponding to this product is detected after incubation in P388-45/C NE (Fig. 5 A, Lanes 3 and 4).

To provide evidence that lack of ESE-dependent splicing in P388-45/C NE was because of the SR proteins phosphorylation level, we complemented P388 and P388-45/C NE with purified recombinant ASF/SF2 expressed in a baculovirus system (bASF/SF2), where the phosphorylation of recombinant proteins is expected to take place. The bASF/SF2 showed an altered migration (Fig. 4,B) entirely attributable to phosphorylation, as judged by phosphatase treatment of the purified protein (data not shown). When assayed with the DUP 3S substrate containing the ASF/SF2 target sequences, complementation of P388-45/C NE with highly phosphorylated bASF/SF2 (Fig. 5,B, Lanes 7 and 8) but not less phosphorylated version (Fig. 5,B, Lanes 9 and 10) restored a splicing activity similar to that of P388 extracts (Fig. 5 B, compare Lanes 1 and 7 and 8). The amount of purified bASF/SF2 added was of the same order of magnitude than that contained in our P388-45/C NE, as judged from Western blot analysis performed with an ASF/SF2 specific antibody (data not shown). A similar quantity of unphosphorylated expressed in E. coli was rather detrimental for splicing (data not shown). Thus, the data support the notion that Topo I-mediated ASF/SF2 complete phosphorylation is critical for ESE-dependent but not constitutive splicing.

Regulation of Alternative Splicing in Topo I-Deficient Cells.

To confirm the alteration of ESE-dependent splicing observed in vitro with P388-45/C extracts, the DUP 3S construct, inserted within a suitable expression vector (24), was transfected in P388 and P388-45/C cells. Total RNA was prepared 24 and 48 h after transfection and the DUP 3S RNA products were analyzed by RT-PCR. As shown in Fig. 6,C, a major band corresponding to exon inclusion was observed in P388 cells (Fig. 6,C, Lane 1) consistent with the role of 3S sequences as powerful and functional ESE in living cells. Failure to detect inclusion of the alternative exon after transfection in P388-45/C cells (Fig. 6 C, Lane 2) confirms the in vitro data and strengthens the correlation between the absence of Topo I and the low levels of enhancer-dependent splicing.

Whether ectopic expression of Topo I could restore normal splicing activity in P388-45/C cells was then assessed after transfection with a GFP-human Topo I expression vector and selection of stably transfected cells in the presence of geneticin. As a control, P388-45/C cells were also transfected with a vector expressing GFP alone and selected as mentioned above. After preparation of NE from both cell populations (45/C-GFP and 45/C-GFP Topo), Western blot analysis performed with anti-Topo I (Fig. 6,A) and anti-GFP mAbs (data not shown) indicated that a GFP-Topo I fusion protein of the expected size is efficiently expressed in 45/C-GFP Topo cells (Fig. 6,A, Lane 4). Two-dimensional Western blot analysis of the corresponding NE with anti-ASF/SF2 mAbs further established that highly phosphorylated variants of this splicing factor are over again detected in 45/C cells expressing the GFP-Topo I fusion protein (Fig. 6 B) but not in 45/C-GFP cells (data not shown).

The 45/C-GFP and 45/C-GFP Topo cells were transiently transfected with the DUP S expression vector, and splicing of the corresponding pre-mRNA was analyzed as described previously. Although spliced forms of the DUP S transcript are not observed in 45/C-GFP cells (Fig. 6,C, Lane 3), the band corresponding to inclusion of the alternative exon in the DUP S mRNA is clearly detected in 45/C-GFP Topo cells (Fig. 6 C, Lane 4). Taken together, these results establish that the DNA Topo I kinase activity is responsible, at least in part, for enhancer-dependent splicing efficiency.

However, ectopic expression of GFP-Topo I was unable to rescue the wild-type expression levels of genes subjected to alternative splicing and/or ESE-dependent splicing (protein kinase C, α actinin 2-associated LIM protein, CD24, and β globin), the expression of which is shut down in P388-45/C cells (Fig. 6 D). This result can be explained if absence of Topo I and/or its inactivation by CPT alters the expression of these genes in such a way that they become stably inactivated through epigenetic modification (32). Consistent with this explanation, treatment of parental cells with CPT dramatically reduces the expression of all above mentioned genes (data not shown).

CPT-resistant cell lines described thus far exhibit either mutations in the Topo I gene, reduced cellular accumulation of CPT, or decreased Topo I expression level (18). CPT-resistant cell lines being Topo I-deficient constitute very useful tools to analyze the consequences of Topo I depletion toward numerous biological processes. In this study, aimed at deciphering the role of the Topo I kinase activity in splicing regulation, we have used the P388-45/C cell line selected for its resistance to 45 μm CPT but which is also able to grow in the presence of 100 μm CPT (data not shown). Western blot analyzes performed with monoclonal and polyclonal antibodies indicate that Topo I is no longer detected in P388-45/C cells (Fig. 1), whereas there is only 4-fold reduction of the corresponding mRNA level in these cells (Table 1). Because sequencing of the Topo I cDNA did not reveal mutations precluding its translation in the resistant cells (data not shown), it is likely that lack of Topo I results from activation of posttranslational degradation mechanisms. Along this line, ubiquitin/26S proteasome-mediated degradation of Topo I has been reported as a resistance mechanism to CPT in tumor cells (33). Another possible explanation comes from the results of our microarray analyzes indicating that P388-45/C cells express elevated levels of mRNA coding for caspase 6 (Table 2, Fig. 2 A), which has been shown to cleave Topo I late during apoptosis (34). In any case, the fact that P388-45/C cells are viable in the absence of detectable Topo I protein demonstrates that, as with yeast (35, 36), Topo I is not required for survival of mammalian cells in culture. However, Topo I is required for embryonic development both in Drosophila (37) and in mice (38).

The results of the Affymetrix analysis strongly suggest that the extinction of Topo I expression is sufficient to overcome the deleterious effects of high CPT concentrations in P388-45/C cells. Indeed, expression of genes such as those encoding multidrug resistance protein 1, P-glycoproteins, and efflux transporters involved in the multidrug resistance phenomenon was not found to be significantly increased in P388-45/C cells, whereas expression of the RAD1 gene, known to be involved in the repair of Topo I-DNA covalent complexes induced by CPT (39), is rather decreased in these resistant cells (Table 1). Moreover, among the genes whose expression was previously found to be increased in HCT116 cells treated with CPT (40), only the one coding for the 14-3-3 ς protein is up-regulated (2.5-fold change). These results indicate that most of the transcriptional changes observed after CPT treatment and corresponding either to primary responses to DNA damage or secondary to cellular processes responding to the damage are not triggered in P388-45/C cells.

Because DNA Topo I is an essential enzyme likely involved in fundamental processes such as DNA replication, recombination, and transcription (Ref. 4) for review), one can wonder that Affymetrix microarray analysis provide clues about how Topo I-deficient cells have survived the lack of such a protein. As already mentioned, progressive up-regulation of genes encoding cytokines and/or their receptors, as well as down-regulation of genes coding for antiproliferative proteins such as Tyk2 (41) or CD24 antigen (42) could account for the survival of P388-45/C cells. However, careful examination of the Affymetrix data indicate that the genes whose expression is induced in Topo I-deficient cells are also linked to apoptosis and growth inhibition (Table 1). This is particularly obvious in the case of genes encoding the interleukin 1 β converting enzyme (caspase 1) involved in the processes of programmed cell death and cytokines activation (Ref. 43 for review), epithelial membrane protein 1 (EMP1/B4B) associated with differentiation, and growth arrest in hematopoietic cells (44), or cyclin G2, the up-regulation of which correlates with cycle cell arrest in B lymphocytes (45). Accordingly, the expression of several genes clearly involved in cellular proliferation, among which those coding for cyclin E (46) and extracellular signal-regulated kinase 5 (47), are down-regulated in Topo I-deficient cells. Taken together, these results indicate that Topo I-deficient cells have developed transcriptional responses not necessarily favoring proliferation processes. The finding that the P388-45/C cells doubling time is approximately twice that of their parental counterparts could reflect the need of a reduced growth rate to preserve DNA and/or RNA integrity in absence of Topo I. This would ultimately reduce susceptibility of P388-45/C cells to DNA damage and contribute to their low sensitivity to CPT. More generally, these results support that in addition to the extent and persistence of Topo I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of cell responsiveness to CPT. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile. Thus, the alterations of gene expression observed in P388-45/C cells may highlight novel targets allowing to increase the efficacy of CPT and other anti-Topo I cancer drugs. The design of novel molecules raised against such targets should prove to be very useful to overcome the processes of cellular resistance to anti-Topo I drugs.

Aside from genes in which the expression has been down-regulated to ensure survival of Topo I-deficient cells, some others were likely identified by the microarray analysis because splicing of the corresponding pre-mRNA is altered in P388-45/C cells. Interestingly, the β-globin gene belongs to the genes whose expression is decreased in P388-45/C cells (Table 1, Fig. 2,B). Indeed, although β-globin is a constitutively spliced pre-mRNA, it contains multiple splicing enhancers within its coding sequences (48). As already seen with the transfected DUP 3S construct (Fig. 6), failure of ESE-dependent splicing in Topo I-deficient cells can lead to intron retention and trigger rapid degradation of the corresponding RNAs, either by activating nonsense-mediated degradation processes (49) or by sequestering incompletely spliced products within the nucleus. Along this line, BLAT analysis of the different mRNA isoforms and expressed sequence tags deposited in European Molecular Biology Laboratory and GenBank databases revealed that >50% of the down-regulated genes identified in this study can undergo alternative splicing (Table 1). Moreover, examining the genomic organization of these genes indicates that almost one-third of them (independently of whether or not they are subjected to alternative splicing) have a large first intron with a size exceeding 5 kb, and their coding sequences split into small exons of <100 nt. An example is the protein kinase C gene in which the first, second, and third introns have a size of 64, 41, and 32 kb, respectively. In contrast, less up-regulated genes (∼20%) display similar features. In light of the studies performed on the β-globin mRNA (50), it is tempting to speculate that accurate removal of large introns from constitutively spliced transcripts rely on the recognition of multiple splicing enhancer elements increasing the probability of an interaction between the splicing machinery and the enhancer complex. Altered ability of SR proteins to interact with their cognate ESE or/and with spliceosome components in Topo I-deficient P388-45/C cells could then account for the observed down-regulation of several genes containing alternative exons or large introns. As already mentioned, the fact that 45/C-GFP Topo cells did not recover wild-type expression levels for such genes raises the interesting possibility that expression of aberrantly spliced mRNAs has triggered epigenetic modifications leading to stable inactivation of these genes.

Study of the splicing events occurring in P388-45/C cells sheds new light on the role of Topo I kinase activity in splicing regulation. In these resistant cells, we have established that the lack of Topo I is correlated to a subtle decrease of SR proteins phosphorylation level (Fig. 3) and to an impairment of ESE-dependent but not constitutive splicing, both in vitro and ex vivo (Fig. 4). These results provide insight in the specificity of Topo I-mediated phosphorylation and its role toward these two splicing mechanisms.

First, the phosphorylation defect of SR proteins clearly established in P388-45/C cells strongly suggests that the absence of Topo I cannot be compensated by the two other classes of kinases, namely SR protein kinases and Cyclin-like kinases (Ref. 9 for review), also known to phosphorylate these splicing regulators. Although we did not check whether expression of these kinases was altered at the protein level in P388-45/C cells, Affymetrix microarray analysis did not reveal significant changes in their mRNA levels (data not shown). Moreover, CPT used as a selection pressure to isolate and maintain the P388-45/C subclone is highly specific for Topo I and does not interfere with SRPK and Clk kinase activity.

The second point relies on the fact that the Topo I deficiency does not significantly affect constitutive splicing but rather inhibits splicing events depending on the recognition of ESEs by SR proteins. Because phosphorylation level has been shown to modulate both the subcellular and subnuclear localization of SR proteins, one could also argue that the Topo I deficiency is correlated to a decrease of SR proteins levels in the nuclear compartment. However, the results of Western blot analyses performed with mAbs specific for different SR factors indicate that their abundance is very similar in NE purified from P388 and P388-45/C cells (data not shown). Moreover, our complementation assays have established that only highly phosphorylated forms of ASF/SF2 are able to restore an ESE-dependent splicing activity in Topo I-deficient extracts. Taken together, these observations indicate that either complete phosphorylation of SR splicing factors or Topo I-mediated phosphorylation at specific sites is required to properly achieve ESE-dependent splicing. Given that phosphorylation of ASF/SF2 has been shown to affect not only its interaction with RNA but also its protein-protein interactions with other SR proteins and constitutive components of the spliceosome, it is tempting to speculate that the role of Topo I-mediated phosphorylation of SR proteins is to promote RNA-protein and/or protein-protein interactions crucial for ESE-dependent but not constitutive splicing events. Targeting the kinase activity of Topo I could therefore offer opportunities to develop novel anticancer drugs interfering with the expression of specific genes involved in cell proliferation and/or apoptosis.

Grant support: Action Concertée Incitative “Molécules et Cibles Thérapeutiques” grant from the French Ministry of Research and a grant from the Association pour la Recherche sur le Cancer (to J. S.). M. G. was supported by a graduate fellowship from the Ministère Délégue à la Recherche et aux Technologies.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J. S. and M. G. contributed equally to this work.

Requests for reprints: Phone: 32-0-467-613-685; Fax: 011-33-4-67-04-02-31; E-mail: tazi@igm.cnrs-mop.fr

4

The abbreviations used are: Topo I, topoisomerase I; SR, serine/arginine-rich; ESE, exonic splicing enhancer; mAb, monoclonal antibody; NE, nuclear extract; CPT, camptothecin; GFP, green fluorescent protein; PM, perfect match oligonucleotide; RT-PCR, reverse transcription-PCR; ASF/SF2, alternative splicing factor/splicing factor 2; SC35, splicing component of 35 kDa.

Fig. 1.

Lack of Topo I in NEs from P388-45/C cells. A, analysis of the DNA relaxation activity in P388 and P388-45/C NE. Assays were performed as described in “Materials and Methods” with 1 μg of supercoiled plasmidic DNA and either 3, 6, or 15 μg of the indicated NE. Control reaction (Ctr) was performed without NE. B, Western blot analysis of Topo I expression levels in P388 and P388-45/C cells. NEs (50 μg) resolved in 10% polyacrylamide-SDS gels and transferred onto nitrocellulose membranes were probed with the indicated antibodies.

Fig. 1.

Lack of Topo I in NEs from P388-45/C cells. A, analysis of the DNA relaxation activity in P388 and P388-45/C NE. Assays were performed as described in “Materials and Methods” with 1 μg of supercoiled plasmidic DNA and either 3, 6, or 15 μg of the indicated NE. Control reaction (Ctr) was performed without NE. B, Western blot analysis of Topo I expression levels in P388 and P388-45/C cells. NEs (50 μg) resolved in 10% polyacrylamide-SDS gels and transferred onto nitrocellulose membranes were probed with the indicated antibodies.

Close modal
Fig. 2.

Confirmation by RT-PCR analysis that expression of several genes, identified by the Affymetrix microarray approach, is indeed either up-regulated (A) or down-regulated (B) in P388-45/C Topo I-deficient cells. Expression of different genes sampled from Tables 1 and 2 was examined by semiquantitative RT-PCR analysis. Expression of the RPL11 gene was used to normalize for amplification efficiency.

Fig. 2.

Confirmation by RT-PCR analysis that expression of several genes, identified by the Affymetrix microarray approach, is indeed either up-regulated (A) or down-regulated (B) in P388-45/C Topo I-deficient cells. Expression of different genes sampled from Tables 1 and 2 was examined by semiquantitative RT-PCR analysis. Expression of the RPL11 gene was used to normalize for amplification efficiency.

Close modal
Fig. 3.

Two-dimensional Western blot analysis of SR protein phosphorylation variants in P388 and P388-45/C cells. A, phosphorylated SR proteins present in the P388 and P388-45/C NE (600 μg/assay) were revealed as a whole with the mAb 104 antibodies. Analysis of the phosphorylation variants corresponding to the individual SR proteins ASF/SF2 (B) and SC35 (C) was performed with the indicated monoclonal antibodies under the same conditions.

Fig. 3.

Two-dimensional Western blot analysis of SR protein phosphorylation variants in P388 and P388-45/C cells. A, phosphorylated SR proteins present in the P388 and P388-45/C NE (600 μg/assay) were revealed as a whole with the mAb 104 antibodies. Analysis of the phosphorylation variants corresponding to the individual SR proteins ASF/SF2 (B) and SC35 (C) was performed with the indicated monoclonal antibodies under the same conditions.

Close modal
Fig. 4.

In vitro ESE-dependent splicing is impaired in NE from P388-45/C cells. NEs (8 μl) purified from P388 (Lanes 1 and 2) or P388–45/C (Lanes 3 and 4) cells were preincubated for 60 (Lanes 1–3) or 40 min (Lanes 2–4) under splicing conditions before addition of the indicated labeled transcript to recycle preassembled complexes. Splicing reaction was then performed at 30°C for 1 h, and splicing products were resolved in a denaturing 7% polyacrylamide gel.

Fig. 4.

In vitro ESE-dependent splicing is impaired in NE from P388-45/C cells. NEs (8 μl) purified from P388 (Lanes 1 and 2) or P388–45/C (Lanes 3 and 4) cells were preincubated for 60 (Lanes 1–3) or 40 min (Lanes 2–4) under splicing conditions before addition of the indicated labeled transcript to recycle preassembled complexes. Splicing reaction was then performed at 30°C for 1 h, and splicing products were resolved in a denaturing 7% polyacrylamide gel.

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Fig. 5.

Restoration of in vitro ESE-dependent splicing by complementation of P388-45/C NE with highly phosphorylated ASF/SF2. A. In vitro splicing of the DUP 3S transcript in 30% (Lanes 1–3) or 40% (Lanes 2–4) of the indicated NE. B, Western blot analysis of recombinant ASF/SF2 proteins phosphorylation level (left panel). Recombinant ASF/SF2 (1.5 μg) produced either in E. coli (Lane 1) or in a baculoviral system (Lanes 2–3) was resolved in a SDS 10% polyacrylamide gel, transferred onto nitrocellulose and probed with anti-ASF/SF2 mAbs. Complementation of P388 and P388-45/C NE with differentially phosphorylated ASF/SF2 recombinant proteins (right panel). Indicated NE (40% final concentration) were complemented with 100 ng (Lanes 2, 4, 7, and 9) or 200 ng (Lanes 3, 5, 8, and 10) of either phosphorylated (Lanes 2, 3, 7, and 8) or highly phosphorylated (Lanes 4, 5, 9, and 10) ASF/SF2 recombinant protein, immediately before addition of the labeled transcript. Reaction was performed at 30°C for 1 h, and splicing products were resolved in a denaturing 7% polyacrylamide gel.

Fig. 5.

Restoration of in vitro ESE-dependent splicing by complementation of P388-45/C NE with highly phosphorylated ASF/SF2. A. In vitro splicing of the DUP 3S transcript in 30% (Lanes 1–3) or 40% (Lanes 2–4) of the indicated NE. B, Western blot analysis of recombinant ASF/SF2 proteins phosphorylation level (left panel). Recombinant ASF/SF2 (1.5 μg) produced either in E. coli (Lane 1) or in a baculoviral system (Lanes 2–3) was resolved in a SDS 10% polyacrylamide gel, transferred onto nitrocellulose and probed with anti-ASF/SF2 mAbs. Complementation of P388 and P388-45/C NE with differentially phosphorylated ASF/SF2 recombinant proteins (right panel). Indicated NE (40% final concentration) were complemented with 100 ng (Lanes 2, 4, 7, and 9) or 200 ng (Lanes 3, 5, 8, and 10) of either phosphorylated (Lanes 2, 3, 7, and 8) or highly phosphorylated (Lanes 4, 5, 9, and 10) ASF/SF2 recombinant protein, immediately before addition of the labeled transcript. Reaction was performed at 30°C for 1 h, and splicing products were resolved in a denaturing 7% polyacrylamide gel.

Close modal
Fig. 6.

Ectopic expression of a GFP-Topo I fusion protein restores ASF/SF2 phosphorylation level and ESE-dependent splicing in P388-45/C cells but not wild-type endogenous expression levels of genes subjected to alternative or ESE-dependent splicing. A, Western blot analysis of endogenous and exogenous Topo I expression levels in NE from nontransfected (Lanes 1 and 2) and transfected cells (Lanes 3 and 4). The NE (100 μg) was probed with monoclonal anti-Topo I antibodies. B, two-dimensional Western blot analysis of ASF/SF2 phosphorylation variants in control (45/C-GFP) and transfected P388-45/C (45/C-GFP Topo) cells. C, RT-PCR analysis of DUP 3S splicing products purified after transient transfection of the P388 (Lane 1), P388-45/C (Lane 2), 45/C-GFP (Lane 3), and 45/C-GFP Topo cells. The structure of the splicing products corresponding to the major bands is depicted on the right. D, RT-PCR analysis of endogenous mRNA expression levels in P388 (Lane 1), P388-45/C (Lane 2), and 45/C-GFP Topo cells (Lane 3). The names of the corresponding genes are indicated.

Fig. 6.

Ectopic expression of a GFP-Topo I fusion protein restores ASF/SF2 phosphorylation level and ESE-dependent splicing in P388-45/C cells but not wild-type endogenous expression levels of genes subjected to alternative or ESE-dependent splicing. A, Western blot analysis of endogenous and exogenous Topo I expression levels in NE from nontransfected (Lanes 1 and 2) and transfected cells (Lanes 3 and 4). The NE (100 μg) was probed with monoclonal anti-Topo I antibodies. B, two-dimensional Western blot analysis of ASF/SF2 phosphorylation variants in control (45/C-GFP) and transfected P388-45/C (45/C-GFP Topo) cells. C, RT-PCR analysis of DUP 3S splicing products purified after transient transfection of the P388 (Lane 1), P388-45/C (Lane 2), 45/C-GFP (Lane 3), and 45/C-GFP Topo cells. The structure of the splicing products corresponding to the major bands is depicted on the right. D, RT-PCR analysis of endogenous mRNA expression levels in P388 (Lane 1), P388-45/C (Lane 2), and 45/C-GFP Topo cells (Lane 3). The names of the corresponding genes are indicated.

Close modal
Table 1

Genes down-regulated in Topo I-deficient cells

Accession no.Fold decreaseAlternatively spliced genes
Immune response genes    
 CD24a antigen M58661 158.00  
 Solute carrier family 12 U13174 68.60 
 Castaneus IgK chain gene, C-region, 3 end M80423 14.10  
 CD3 antigen, γ polypeptide M18228 13.00 
 Rearranged immunoglobulin γ 2b heavy chain X67210 12.20 
 Histocompatibility 2, class II antigen A, α X52643 11.00 
 Ig κ chain V-region mRNA M18237 10.70 
 Anti-PAH immunoglobulin Fab 4D5 U62650 7.85 
 Anti-HIV-1 reverse transcriptase U48716 7.50  
 Lymphoid-specific interferon regulatory factor (LSIRF) U20949 7.25  
 Ig-kappa light chain V-J κ 5 joining region X00651 7.00 
 Melanoma antigen D10049 6.00 n.d.a 
 Fc receptor, IgG X70980 5.90  
 Immunoglobulin kappa light chain (V κ) AF045026 5.20 
 Major histocompatibility locus class III AF109905 5.30  
Genes involved in proliferation, differentiation, and signalling    
 Early growth response 1 M28845 14.30 
 Receptor activity modifying protein AJ250489 10.30  
 Cyclin E X75888 10.10 
 Rho-GDI2 U73198 10.00  
 Insulin-like growth factor 2 X71922 9.10  
 Somatostatin receptor 3 M91000 8.40  
 XMR X72697 8.35  
 Erythroid differentiation regulator AJ007909 8.70  
 MAST205 protein kinase mRNA U02313 7.20  
 ERK5 AB019373 6.90 
 Receptor tyrosine kinase AB000828 6.50  
 Tyk2 AF052607 5.50 
 Cyclin-dependent kinase 5 D29678 5.40 
 Fast2 AF069303 5.40  
 Epimorphin D10475 5.40  
 Protein kinase C D90242 5.30 
 SseCKs AB020886 4.60  
DNA binding proteins, transcription, and splicing factors    
 Inhibitor of DNA binding 3 M60523 16.60 
 Histone 1 M29260 16.20 n.d. 
 Ets transcription factor SpiB U87620 14.80 
 SAP62-AMH X83733 11.20 
 RAD1 (Rad1) AF073523 7.10 
 Zinc-finger protein U14556 4.60 
 Histone homologue A X92490 4.55 
 Sine occulis-related D83146 4.50  
 TOP gene for Topo I X70956 4.45  
 Nuclear transcription factor-Y γ U62297 4.35  
 Transcription factor P113 AF010138 3.80 
Structural proteins    
 Lamin A D49733 16.70 
 Hair keratin acidic 5 AF020790 12.00  
 Neuronal adhesion molecule AF026465 8.50  
 High-sulfur keratin protein D86424 7.80 n.d. 
 SRG3 mRNA U85614 6.20  
 Vascular smooth muscle α-actin X13297 5.80 
 Phospholipid transfer protein U28960 5.10 
 Apolipoprotein E D00466 4.70  
 Epidermal keratin J02644 4.25  
Metabolic enzymes    
 Balb/c cytochrome c oxidase subunit VIaH U08439 41.20 
 Stearoyl-coenzyme A desaturase 1 M21285 40.50 
 Glucosidase, α, acid U49351 17.10 
 Microsomal expoxide hydrolase (Eph1) U89491 10.00 
 Mast cell protease 5 M68898 8.10  
 Adenine phosphoribosyl transferase M11310 7.90 
 Glyceraldehyde 3-phosphate dehydrogenase U09964 7.30 
 PAF acetylhydrolase mRNA U34277 7.25 
 Kallikrein X03994 5.50 
 Hydroxymethyltransferase X94478 4.80  
 Adrenodoxin L29123 4.70  
 Guanidinoacetate methyltransferase AF010499 3.90 
 Amine oxidase, copper containing 3 AF078705 3.80 
Miscellaneous genes    
 Hemoglobin, β adult major chain J00413 11.50 
 β-1-globin V00722 6.45 
 DMR-N9 Z38011 6.40 
 Transition protein 2 M60254 5.60  
 Periferin 2 X14770 4.65  
 Somatostatin X51468 4.40  
Accession no.Fold decreaseAlternatively spliced genes
Immune response genes    
 CD24a antigen M58661 158.00  
 Solute carrier family 12 U13174 68.60 
 Castaneus IgK chain gene, C-region, 3 end M80423 14.10  
 CD3 antigen, γ polypeptide M18228 13.00 
 Rearranged immunoglobulin γ 2b heavy chain X67210 12.20 
 Histocompatibility 2, class II antigen A, α X52643 11.00 
 Ig κ chain V-region mRNA M18237 10.70 
 Anti-PAH immunoglobulin Fab 4D5 U62650 7.85 
 Anti-HIV-1 reverse transcriptase U48716 7.50  
 Lymphoid-specific interferon regulatory factor (LSIRF) U20949 7.25  
 Ig-kappa light chain V-J κ 5 joining region X00651 7.00 
 Melanoma antigen D10049 6.00 n.d.a 
 Fc receptor, IgG X70980 5.90  
 Immunoglobulin kappa light chain (V κ) AF045026 5.20 
 Major histocompatibility locus class III AF109905 5.30  
Genes involved in proliferation, differentiation, and signalling    
 Early growth response 1 M28845 14.30 
 Receptor activity modifying protein AJ250489 10.30  
 Cyclin E X75888 10.10 
 Rho-GDI2 U73198 10.00  
 Insulin-like growth factor 2 X71922 9.10  
 Somatostatin receptor 3 M91000 8.40  
 XMR X72697 8.35  
 Erythroid differentiation regulator AJ007909 8.70  
 MAST205 protein kinase mRNA U02313 7.20  
 ERK5 AB019373 6.90 
 Receptor tyrosine kinase AB000828 6.50  
 Tyk2 AF052607 5.50 
 Cyclin-dependent kinase 5 D29678 5.40 
 Fast2 AF069303 5.40  
 Epimorphin D10475 5.40  
 Protein kinase C D90242 5.30 
 SseCKs AB020886 4.60  
DNA binding proteins, transcription, and splicing factors    
 Inhibitor of DNA binding 3 M60523 16.60 
 Histone 1 M29260 16.20 n.d. 
 Ets transcription factor SpiB U87620 14.80 
 SAP62-AMH X83733 11.20 
 RAD1 (Rad1) AF073523 7.10 
 Zinc-finger protein U14556 4.60 
 Histone homologue A X92490 4.55 
 Sine occulis-related D83146 4.50  
 TOP gene for Topo I X70956 4.45  
 Nuclear transcription factor-Y γ U62297 4.35  
 Transcription factor P113 AF010138 3.80 
Structural proteins    
 Lamin A D49733 16.70 
 Hair keratin acidic 5 AF020790 12.00  
 Neuronal adhesion molecule AF026465 8.50  
 High-sulfur keratin protein D86424 7.80 n.d. 
 SRG3 mRNA U85614 6.20  
 Vascular smooth muscle α-actin X13297 5.80 
 Phospholipid transfer protein U28960 5.10 
 Apolipoprotein E D00466 4.70  
 Epidermal keratin J02644 4.25  
Metabolic enzymes    
 Balb/c cytochrome c oxidase subunit VIaH U08439 41.20 
 Stearoyl-coenzyme A desaturase 1 M21285 40.50 
 Glucosidase, α, acid U49351 17.10 
 Microsomal expoxide hydrolase (Eph1) U89491 10.00 
 Mast cell protease 5 M68898 8.10  
 Adenine phosphoribosyl transferase M11310 7.90 
 Glyceraldehyde 3-phosphate dehydrogenase U09964 7.30 
 PAF acetylhydrolase mRNA U34277 7.25 
 Kallikrein X03994 5.50 
 Hydroxymethyltransferase X94478 4.80  
 Adrenodoxin L29123 4.70  
 Guanidinoacetate methyltransferase AF010499 3.90 
 Amine oxidase, copper containing 3 AF078705 3.80 
Miscellaneous genes    
 Hemoglobin, β adult major chain J00413 11.50 
 β-1-globin V00722 6.45 
 DMR-N9 Z38011 6.40 
 Transition protein 2 M60254 5.60  
 Periferin 2 X14770 4.65  
 Somatostatin X51468 4.40  
a

n.d., not determined.

Table 2

Genes up-regulated in Topo I-deficient cells

Accession no.Fold inductionAlternatively spliced genes
Immune response genes    
 Ly-6 alloantigen (Ly-6E.1) X04653 62.75  
 Small inducible cytokine A9 U49513 30.6  
 CD2 antigen X06143 29  
 Caspase-6 Y13087 12.66  
 Interleukin 1 receptor, type II X59769 12.55  
 Tumor necrosis factor (ligand) AF019048 11.95 
 Transforming growth factor, β 3 M32745 13.5  
 CD5 antigen M15177 10.15  
 MIP-1b X62502 9.75  
 pM1 protein X07967 8.85  
 Cytotoxic T lymphocyte-associated protein 2 X15591 7.4  
 Interferon (α and β) receptor AF030311 6.2  
 Lymphocyte antigen 9 M84412 6.1  
 FCRII M31312 6.05 
 CRTAM AF001104 5.6  
 Ig Vκ-HNK20 X82688 5.5  
 Interleukin 1-β converting enzyme L28095 5.05  
 Histocompatibility 2 Y00629 4.65  
 p75 TNF receptor DNA X87128 4.4  
Genes involved in proliferation, differentiation, and signalling    
 Epithelial membrane protein 1 X98471 27.7  
 Granuphilin-a AB025258 15.5 
 Granuphilin-b AB025259 14.05 
 G-protein-like LRG-47 U19119 8.95  
 TDD5 mRNA U52073 9.85 
 Thromboxane A2 receptor D10849 7.7  
 Cyclin G2 mRNA U95826 6.15  
 Apoptosis activator Mtd (Mtd) mRNA AF027707 6.1  
 76 kDa tyrosine phosphoprotein U20159 6.75  
 IRG-47 M63630 4.85 
 Calcium-binding protein A4 M36579 4.8 
 SH3 binding protein 3BP2 mRNA L14543 4.75  
 Protein tyrosine phosphatase epsilon D83484 4.55 
 Calcium-binding protein A13 X99921 4.25  
 Insulin like growth factor binding protein 4 X76066 3.9  
 α diacylglycerol kinase mRNA AF085219 3.7  
DNA binding proteins and transcription factors    
 Max-interacting transcriptional repressor (Mad4) mRNA U32395 19.5  
 Inhibitor of DNA binding 2 AF077861 5.2  
 Transcription factor GIF mRNA AF064088 4.5  
 Recombination-activating gene 1 M29475 3.5  
Structural proteins    
 Myosin heavy chain, cardiac muscle M76599 10.3  
 Myosin VIIa U81453 8.8 
 Latexin D88769 6.5  
 Myelin basic protein M11533 6.45 
 Amyloid precursor protein (APP) gene U82624 6.20 
 Slow myosin heavy chain-β AJ223362 5.25  
 Myosin X AJ249706 4.50  
Metabolic enzymes    
 Serine protease hepsin mRNA AF030065 23.4  
 Glutathione S-transferase, θ 2 X98056 16.8  
 Aldo-keto reductase AKR1C13 AB027125 13.1  
 Leukotriene C4 synthase U27195 7.25  
 Cathepsin E AJ009840 4.70  
 Arginase II AF032466 4.6  
 PAF-AH γ U57746 4.3  
 Malic enzyme J02652 4.05 
Miscellaneous genes    
 Cytoplasmic protein Ndr1 (Ndr1) mRNA U60593 11 
 Placentae and embryos oncofetal M32484 11  
 Properdin factor X12905 8.50  
 Presynaptic protein D38614 7.50  
 P50p D49691 7.40  
 Centromere autoantigen B X55038 5.60  
 Topoisomerase-inhibitor suppressed D86344 5.35  
 Biliary glycoprotein 1 M77196 
 Protective protein for β-galactosidase J05261 3.9  
Accession no.Fold inductionAlternatively spliced genes
Immune response genes    
 Ly-6 alloantigen (Ly-6E.1) X04653 62.75  
 Small inducible cytokine A9 U49513 30.6  
 CD2 antigen X06143 29  
 Caspase-6 Y13087 12.66  
 Interleukin 1 receptor, type II X59769 12.55  
 Tumor necrosis factor (ligand) AF019048 11.95 
 Transforming growth factor, β 3 M32745 13.5  
 CD5 antigen M15177 10.15  
 MIP-1b X62502 9.75  
 pM1 protein X07967 8.85  
 Cytotoxic T lymphocyte-associated protein 2 X15591 7.4  
 Interferon (α and β) receptor AF030311 6.2  
 Lymphocyte antigen 9 M84412 6.1  
 FCRII M31312 6.05 
 CRTAM AF001104 5.6  
 Ig Vκ-HNK20 X82688 5.5  
 Interleukin 1-β converting enzyme L28095 5.05  
 Histocompatibility 2 Y00629 4.65  
 p75 TNF receptor DNA X87128 4.4  
Genes involved in proliferation, differentiation, and signalling    
 Epithelial membrane protein 1 X98471 27.7  
 Granuphilin-a AB025258 15.5 
 Granuphilin-b AB025259 14.05 
 G-protein-like LRG-47 U19119 8.95  
 TDD5 mRNA U52073 9.85 
 Thromboxane A2 receptor D10849 7.7  
 Cyclin G2 mRNA U95826 6.15  
 Apoptosis activator Mtd (Mtd) mRNA AF027707 6.1  
 76 kDa tyrosine phosphoprotein U20159 6.75  
 IRG-47 M63630 4.85 
 Calcium-binding protein A4 M36579 4.8 
 SH3 binding protein 3BP2 mRNA L14543 4.75  
 Protein tyrosine phosphatase epsilon D83484 4.55 
 Calcium-binding protein A13 X99921 4.25  
 Insulin like growth factor binding protein 4 X76066 3.9  
 α diacylglycerol kinase mRNA AF085219 3.7  
DNA binding proteins and transcription factors    
 Max-interacting transcriptional repressor (Mad4) mRNA U32395 19.5  
 Inhibitor of DNA binding 2 AF077861 5.2  
 Transcription factor GIF mRNA AF064088 4.5  
 Recombination-activating gene 1 M29475 3.5  
Structural proteins    
 Myosin heavy chain, cardiac muscle M76599 10.3  
 Myosin VIIa U81453 8.8 
 Latexin D88769 6.5  
 Myelin basic protein M11533 6.45 
 Amyloid precursor protein (APP) gene U82624 6.20 
 Slow myosin heavy chain-β AJ223362 5.25  
 Myosin X AJ249706 4.50  
Metabolic enzymes    
 Serine protease hepsin mRNA AF030065 23.4  
 Glutathione S-transferase, θ 2 X98056 16.8  
 Aldo-keto reductase AKR1C13 AB027125 13.1  
 Leukotriene C4 synthase U27195 7.25  
 Cathepsin E AJ009840 4.70  
 Arginase II AF032466 4.6  
 PAF-AH γ U57746 4.3  
 Malic enzyme J02652 4.05 
Miscellaneous genes    
 Cytoplasmic protein Ndr1 (Ndr1) mRNA U60593 11 
 Placentae and embryos oncofetal M32484 11  
 Properdin factor X12905 8.50  
 Presynaptic protein D38614 7.50  
 P50p D49691 7.40  
 Centromere autoantigen B X55038 5.60  
 Topoisomerase-inhibitor suppressed D86344 5.35  
 Biliary glycoprotein 1 M77196 
 Protective protein for β-galactosidase J05261 3.9  

We thank Drs. Joao Ferreira and Soren Steffensen for kindly providing us with the GFP-Topo I expression vector. We also thank Frédéric Diemunsch and Christelle Thibault from Affymetrix Platform of the Génopole Alsace-Lorraine for the Affymetrix analysis.

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