Báo cáo khoa học: The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides

Eur. J. Biochem. 269, 583–592 (2002) (cid:211) FEBS 2002

The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides

Anneloor L. M. A. ten Asbroek, Marjon van Groenigen, Marleen Nooij and Frank Baas

Neurozintuigen Laboratory, Academic Medical Center, Amsterdam, the Netherlands

expressed the protein throughout the cell. Whole cell extracts of the cell lines yielded similar RNase H cleavage activity in an in vitro liquid assay, in contrast to the e(cid:129)cacy of the ODNs in vivo. Overexpression of RNase H2 did not a(cid:128)ect the response to ODNs in vivo. Our data imply that in vivo RNase H activity is not only due to the activity assayed in vitro, but also to an intrinsic property of the cells. RNase H1 is not likely to be a major player in the antisense ODN-mediated degradation of target mRNAs. RNase H2 is involved in the activity assayed in vitro. The presence of cell-type specific factors a(cid:128)ecting the activity and localization of RNase H2 is strongly suggested.

Keywords: ribonuclease; RNase H; human; antisense; phosphorothioate.

We have analyzed the response of a number of human cell lines to treatment with antisense oligodeoxynucleotides (ODNs) directed against RNA polymerase II, replication protein A, and Ha-ras. ODN-delivery to the cells was liposome-mediated or via electroporation, which resulted in di(cid:128)erent intracellular locations of the ODNs. The ODN-mediated target mRNA reduction varied consider- ably between the cell lines. In view of the essential role of RNase H activity in this response, RNase H was ana- lyzed. The mRNA levels of RNase H1 and RNase H2 varied considerably in the cell lines examined in this study. The intracellular localization of the enzymes, assayed by green-fluorescent protein fusions, showed that RNase H1 was present throughout the whole cell for all cell types analyzed, whereas RNase H2 was restricted to the nucleus in all cells except the prostate cancer line 15PC3 that

ular mass (30–45 kDa), are activated only by Mg2+ and inhibited by additional Mn2+, and are highly sensitive to sulfhydryl-blocking reagents.

Ribonucleases H (RNases H) are enzymes that specifically hydrolyze the RNA moiety in RNA–DNA duplexes [1,2]. Proteins with RNase H activity are ubiquitous and have been isolated from a variety of organisms, ranging from viruses to prokaryotes and eukaryotes [3]. The best char- acterized and functionally understood RNases H are the RNase H domains of retroviral reverse transcriptases, and the evolutionary related RNase HI of Escherichia coli. For both these enzymes, the crystal structures are available [4,5] and amino-acid residues involved in substrate binding, metal binding, and catalysis have been identified and studied in detail by site-directed mutagenesis [6,7]. Mammalian RNase H enzyme activities have been biochemically char- acterized in various tissues, including calf thymus [8], mouse cells [9], HeLa cells [10], human placenta [11] and human erythroleukemia cells [12]. Based on differences in their biochemical characteristics and immunological cross- reactivity, RNase H activity in higher eukaryotes has been grouped into two classes [13,14]. Class I enzymes have a native molecular mass of 68–90 kDa, are activated by both Mg2+ and Mn2+, and are active in the presence of sulfhydryl reagents. Class II enzymes have a lower molec-

Two different RNases H have been cloned and char- acterized in E. coli: RNase HI [15] and RNase HII [16]. The human sequence homologues of these bacterial enzymes have recently been identified and characterized [17–21]. This has helped to link the biochemically characterized enzyme activities to the gene sequences. An overview of the two RNase H families, and their homologues identified in various species is given by Ohtani et al. [22]. The human RNase H1 is a class I enzyme, and the sequence homologue of E. coli RNase HII, a prokaryotic minor enzyme which is not well characterized. Human RNase H2 is a class II enzyme, and the sequence homologue of E. coli RNase HI, the prokaryotic major enzyme that has been characterized in detail. RNase H enzymes are involved in removing RNA primers in prokaryotic and eukaryotic DNA synthesis reconstitution experiments in vitro [23,24]. The physiological role of RNase HI in E. coli, however, is to prevent replication taking place from sites other than oriC. The RNA primer removal during replication in vivo is performed by the 5¢-exonuclease activity of DNA polymerase I [25]. Similarly, the removal of Okazaki RNA primers in vivo in eukaryotic cells does not necessarily involve RNase H; Dna2 helicase, helicase E, or Ku helicase, acting together with FEN1/RTH1 are also good and possible candidates [26]. The physiological role of the eukaryotic RNases H remains, as yet, undetermined.

The RNases H have gained renewed attention since the development of antisense drugs. Antisense oligodeoxy- nucleotides (ODNs) are widely used as a tool to down- regulate gene expression in a sequence-specific manner. The

Correspondence to F. Baas, Neurozintuigen Laboratory, Academic Medical Center, PO Box22700, 1000 DE Amsterdam, the Netherlands. Fax: + 31 20 5664440, Tel.: + 31 20 5665998, E-mail: f.baas@amc.uva.nl Abbreviations: ODN, oligodeoxynucleotide; PS, phosphorothioate; PO, phosphodiester; RNase, ribonuclease; FITC, fluorescein; GFP, green-fluorescent protein. (Received 13 July 2001, revised 16 November 2001, accepted 17 November 2001)

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complete culture medium and plated at appropriate density for recovery.

single-stranded DNA sequence binds to the complementary site in the target mRNA, upon which the RNA strand of the resulting DNA–RNA hybrid is cleaved by RNase H [27]. Regular phosphodiester (PO) ODNs are rapidly degraded by cellular nucleases, and therefore modified ODNs must be used. Phosphorothioate (PS) ODNs, in which a sulfur atom has replaced the nonbridging oxygen atom of the phosphate backbone, are most often used in practice. They are highly resistant to nucleases, able to recruit RNase H cleavage, and commercially available. Apart from their sequence-specific effects, however, these molecules also exhibit a number of sequence-independent artefacts, most of which are attrib- utable to their ability to bind a number of heparin-binding proteins [28].

Plasmid transfections for transient expression of GFP- constructs were with 2 lg supercoiled plasmid on 105 cells. For fluorescence microscopy, cells were plated on glass coverslips in a six-well culture plate, and transfected with FITC-labeled ODNs or GFP-expressing plasmids. For analysis, cells were fixed on the glass in NaCl/Pi containing 4% paraformaldehyde and embedded in Vectashield Mounting Medium (Vector Laboratories Inc.). Fluor- escence microscopy was carried out with a Vanox micro- scope and appropriate filters. For stable expression of RNase H2 in HEK293, cells were plated in 10-cm dishes at 107 cells and transfected for 5 h in 2.5 mL serum-free medium containing 12.5 lL transfection reagent DAC-30 (Eurogentec) and 2 lg linearized plasmid. Initial selection of transfected cells was with 1.5 mg G418 (Roche) per mL of medium. Maintenance of selected clones was at 0.5 mg G418 per mL.

Tritium ODN measurements

In our search for allele-specific inhibitors based on single- nucleotide polymorphisms in target mRNA sequences using antisense PS-ODNs, which could provide a tumor cell specific anticancer therapy [29], we encountered large differences in the responses of the various human cancer cell lines to the same ODN. We have examined this effect in detail and extended the analysis to different target sequences and ODN delivery methods. Furthermore, we investigated the role of RNase H2 in this process using in vitro and in vivo measurements.

M A T E R I A L S A N D M E T H O D S

Cell culture

Tritium labeling of the ODN was performed using the heat exchange method described by Graham et al. [33]. Cells were transfected with 3H-labeled PS-ODN (specific activity 40 260 d.p.m.Ælg)1 ODN) using the liposomal or electro- poration delivery described above and seeded in six-well plates. At sampling, cells were extensively washed with NaCl/Pi (5 · 3 mL NaCl/Pi per well) and lysed sub- sequently in 1 mL 1 M NaOH per well. Aliquots of 500 lL were used for liquid scintillation counting. Protein concen- tration was measured with Bio-Rad DC reagent (Bio-Rad) using a BSA standard series for quantification.

Plasmids

lines HEK293 (embryonal kidney), 15PC3 Human cell (prostate cancer), MiaPacaII (pancreatic carcinoma), T24 (bladder carcinoma), HeLa (cervical carcinoma) and HTB82 (rhabdomyosarcoma), were obtained from the American Type Culture Collection, or were gifts from colleagues. Cells were maintained by serial passage in Dulbecco’s modified Eagle’s medium (DMEM), supple- mented with 10% fetal bovine serum, 2 mM L-glutamine, 100 UÆmL)1 penicillin, and 100 lgÆmL)1 streptomycin.

Transfections

C-Terminal GFP fusion vector pEGFP-C1 was obtained from Clontech; pcDNA3 was obtained from Invitrogen. pcDNA3-derived constructs were linearized with restriction endonuclease PvuI (Roche) prior to transfection. Coding regions of RNase H1 (GenBank accession no. Z97029) and RNase H2 (GenBank accession no. AF039652) were cloned into pEGFP-C1 or pcDNA3 via RT-PCR with proofreading Taq polymerase (primer sequences available upon request). Constructs used for expression experiments were verified by DNA sequencing using Big-Dye terminator chemistry (PerkinElmer) and analyzed on an ABI377 sequencer.

RNA analysis

Oligonucleotides were purchased from Isogen (the Netherlands). ODNs directed against POLR2A have been described previously [29]. Basilion et al. [30] and Monia et al. [31] have described ODNs ISIS12790 and ISIS 2503 directed against RPA70 and Ha-ras, respectively. ODN transfection with liposomal transfection reagent DAC-30 (Eurogentec) was as described previously and performed in a six-well culture plate, with 1 mL of serum-free medium containing DAC-30 and ODN [29]. ODN transfection by electroporation was carried with a Bio-Rad Gene Pulser II with RF module. One day prior to transfection, cells were plated such that at transfection (cid:25) 70% confluency was reached. Cells were harvested using trypsine followed by washing in NaCl/Pi, and resuspended in Hepes-buffered media (2 mM Hepes, 15 mM K-phosphate buffer, 250 mM [32]) at 106 cells per mannitol, 1 mM MgCl2, pH 7.2; 100 lL. This was incubated with the ODN at ice for 10 min, transferred to an electroporation cuvet (0.2 cm; Bio-Rad) and shocked (280 V, 100% modulation, 140 amplitude, 40 kHz RF, 1.5 ms burst duration, 15 bursts, 1.5 s interval). The cuvet was placed on ice immediately after electroporation. Cells were washed out of the cuvet in

Northern blot analysis of RNA was carried out as described previously [29]. Hybridized probe was visual- ized and quantified on a PhosphoImager (Molecular Dynamics). cDNA fragments to be used as probe were generated by RT-PCR and subsequent cloning into the pGEM-T Easy vector (Promega). Probes used were POLR2A (GenBank accession no. X63564, position 1608–2078), RPA70 (GenBank accession no. M63488, position 1066–1718), Ha-ras (GenBank accession no. J00277, position 1659–3485 exon sequences only), 28S rRNA (GenBank accession no. M11167, position 1635– 1973), and GAPDH (GenBank accession no. M33197, position 245–536).

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RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 585

InvitroRNase H assay

The in vitro RNase H assay is a combination of two protocols described in literature [34,35]. Whole cell extracts were prepared as follows: exponentially growing cells were harvested by scraping, washed once in NaCl/Pi, and resuspended in 100 lL hypotonic lysis buffer (7 mM Tris/ HCl pH 7.5, 7 mM KCl, 1 mM MgCl2, 1 mM 2-mercapto- ethanol) per 106 cells. After 10 min incubation on ice, DNA was sheared by repeated passaging through a 27 Gauge needle. Then, 0.1 vol. of neutralization buffer (21 mM Tris/ HCl pH 7.5, 116 mM KCl, 3.6 mM MgCl2, 6 mM 2-mercaptoethanol) was added. Cell debris was removed by centrifugation for 10 min at 4 (cid:176)C. The supernatant was transferred to a fresh tube on ice and glycerol was added to a final concentration of 45%. The RNase H activity in these extracts is relatively labile and susceptible to freezing or diluting of the extracts. The extracts used in one experiment were always isolated at the same time and treated in the same way. So within one experiment, the ratio of the extracts of different cell lines has to be compared. Absolute levels differ between the experiments. Template RNA was prepared by in vitro transcription of linearized target plasmid construct, using T7 RNA polymerase (Promega) and the manufacturer’s protocol. Run-off RNA and complementary ODN were denatured separately by boiling for 5 min in 100 mM KCl, 0.1 mM EDTA and slowly cooled to room temperature to allow folding of the template. Template RNA (50 ng) and 100 ng ODN were annealed at 37 (cid:176)C for 15 min in 30 lL 100 mM KCl, 0.1 mM EDTA. Then, RNase H mixture was added, comprised of 8.4 lL 5 · buffer (250 mM Tris/HCl pH 7.5, 50 mM MgCl2, 1 mM dithiothreitol, 2.5 mgÆmL)1 BSA), 1 lL RNasin (20 UÆlL)1; Promega) and 5 lL cell extract, and incubated at 37 (cid:176)C for 5 min. RNA was subsequently precipitated in the presence of glycogen, after removal of proteins by phenol extraction, and dissolved in gel loading buffer containing 95% forma- mide. Fragments were separated on a denaturing gel (6% acrylamide, 8 M urea), electroblotted onto Hybond-N+ membrane (Amersham), and visualized by hybridization with a probe derived from the insert of the plasmid used for run-off RNA synthesis.

Fig. 1. Northern blot analysis of RNases H in the cell lines. Total RNA isolated from exponentially growing cells was hybridized to probes for RNase H1 (top) and RNase H2 (middle). The arrow in the middle panel indicates the 1.2-kb main RNase H2 mRNA; the asterisk indi- cates a 5.5-kb RNase H2 mRNA species. The bottom panel shows the 28S rRNA control hybridization.

R E S U L T S

intensity between the two groups, after normalization for 28S rRNA signal, is about 10-fold. The RNase H2 mRNA level shows a fivefold to 10-fold variation, but with a different distribution over the cell lines. 15PC3 and Mia- PacaII display the highest level of the 1.2-kb mRNA, and HEK293 the lowest. The 5.5-kb mRNA species detected by the RNase H2 probe (described by Wu et al. [20] to be a polyadenylated processing variant of the main 1.2-kb mRNA) shows a more or less consistent level in the various cell lines (variation is only up to twofold). Our subsequent analysis focused on the three cell lines that present the possible variation in mRNA levels: MiaPacaII (low RNase H1, high RNase H2), HEK293 (high RNase H1, low RNase H2) and 15PC3 (high RNase H1, high RNase H2).

Six human cell lines (embryonal kidney HEK293, prostate cancer 15PC3, pancreatic carcinoma MiaPacaII, cervical carcinoma HeLa, bladder carcinoma T24, and rhabdomyo- sarcoma HTB82) were analyzed for their response to treatment with antisense ODNs. The initial experiments were performed using liposomal delivery of various anti- sense phosphorothioate ODNs. The response to ODN treatment varied considerably. 15PC3 and MiaPacaII showed a good response, while HEK293 and HTB82 hardly responded at all, and HeLa and T24 showed an intermediate response. To investigate the nature of the differences in response to antisense ODNs we analyzed the RNase H levels in the cell lines, as RNase H is claimed to be a key component in the mechanism of inhibition of gene expres- sion by antisense ODNs. The variation in RNase H mRNA levels is substantial (Fig. 1). HEK293, HeLa and 15PC3 display a similar high level of RNase H1, whereas MiaP- acaII, T24 and HTB82 show a low level. The difference in

As mRNA levels do not necessarily reflect protein levels or activity, we measured the RNase H activity in an in vitro assay using whole cell extracts. An in vitro synthesized run- off RNA, corresponding to a part of the POLR2A mRNA sequence (GenBank accession no. X63564, position 2846– 3306) was hybridized with a complementary phosphodiester (PO) ODN of 16 nucleotides (L5Cas16; position 3049– 3064). Cellular extracts were used in a concentration series to assay the nonsaturated part of the activity curve, and mixtures of two different cell extracts were compared to the

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active as the individual extracts demonstrates that both cell lines harbor similar RNase H enzyme activity. Similar results were obtained with extracts of 15PC3 cells (not shown). Phosphorothioate (PS) ODNs behave similarly to PO-ODNs in the in vitro assay. They are slightly less efficient, yielding 50–60% cleavage of the target RNA with 1 lL extract, compared to 60–70% cleavage using the corresponding PO-ODN (unpublished results).

The in vivo performance of the cells to antisense ODN treatment was tested by transfection experiments. Antisense inhibition of gene expression is presumed to result in degradation of the target mRNA via RNase H activity. The efficacy of a particular ODN can therefore best be addressed by Northern blot analysis of the target mRNA, as the level of full-length mRNA can be assayed. To avoid scoring possible artefacts of the ODN delivery system and chem- istry-related toxicity, we used liposomal delivery of PS-ODNs to the cells (PO-ODNs do not enter the cells via liposomes; A. L. M. A. ten Asbroek unpublished observations) as well as delivery of PS- and PO-ODNs by electroporation. Figure 3A shows the effect of 20 h

separate extracts. A representative example of an RNase H assay is shown in Fig. 2 (Fig. 2A shows the results for HEK293 and Fig. 2B for MiaPacaII). Ten microlitres or 20 lL of extract yields the saturation level of substrate digestion by RNase H in the extracts. Roughly 10% of the input RNA remains uncut. In both cases, the range from 0.5 to 2 lL extract is not yet saturating, indicating a similar level of activity in both cells. Perhaps we measure two distinct activities in these extracts, e.g. RNase H1 in HEK293 and RNase H2 in MiaPacaII, which may be additive or for which one may be limiting. In order to exclude this possibility, equal amounts of both extracts were mixed and compared to the activity of one single extract. Figure 2A shows that 0.5 lL HEK293 extract plus 0.5 lL MiaPacaII extract leads to 76% digestion of the input target RNA, whereas 1 lL extract of HEK293 gives 71% digestion. Similarly, 1 lL of both extracts combined vs. 2 lL of single extract gives 81 vs. 82% digestion, respect- ively. The same is demonstrated in Fig. 2B, where the comparison of combined extracts to single MiaPacaII extract is made. The difference in activity obtained with the combined extracts in Fig. 2A,B reflects the interexperi- mental variation. The fact that the combined extracts are as

Fig. 3. Northern blot analysis of the cell lines transfected with 800 nM ODNs directed against RPA70 and Ha-ras or POLR2A. Probes used are indicated on the left side. 28S rRNA and GAPDH hybridization were used for quantification of RNA loading. ODNs used are indicated on top of the lanes. (A) Liposomal transfection of PS-ODNs: aRPA, ISIS12790 directed against RPA70; aRAS, ISIS2503 directed against Ha-ras; aPOL, L5Cas20 (for 15PC3 and HEK293) or L5Tas20 (for MiaPacaII) directed against POLR2A; 20-mer, completely randomized control mixture of 20-mer PS-ODNs; mock, transfection without PS- ODN. RNA was isolated for analysis at 20 h post-transfection (B) Electroporation transfection of 800 nM PS-ODN ISIS 12790 (RPA-S) and the PO version of this ODN (RPA-O). RNA was isolated for analysis at 4 h or 20 h post-transfection as indicated on the right. Fig. 2. In vitro RNase H assay with whole cell extracts of cell lines HEK293 (A) and MiaPacaII (B). The amount of extract (XT) used is indicated on top of the lanes. The lanes depicted 0.5 + 0.5 and 1 + 1 are assayed with a mixture of both cell extracts. Digested product is detected as a single band on these gels, as the ODN hybridizes to the center of the input target RNA. The asterisk indicates the input target RNA; the arrow indicates the digested product bands. The amount of digested product obtained is indicated at the bottom of the lanes as percentage of total signal detected in the lane (remaining uncut input RNA plus digested product RNA).

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RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 587

Table 1. Percentage of intact target mRNA after antisense ODN treatment. After treatment with 800 nM antisense ODNs, phosphorothioate (POL-S and RPA-S) or phosphodiester (RPA-O), cells were assayed for intact target mRNA at 20 h or 4 h post-transfection, using Northern blotting. Percentages, corrected for loading and normalized to the mock control transfections, are given as mean (cid:139) SD for n independent experiments. ND, not determined; NA, not available, as PO-ODNs do not enter cells when delivered by liposomal transfection reagents.

Delivery system

Sample Liposomal 20 h Electroporation 20 h Electroporation 4 h

19.7 (cid:139) 3.3 (n (cid:136) 6) 26.0 (cid:139) 2.2 (n (cid:136) 3) NA ND 80.7 (cid:139) 9.0 (n (cid:136) 3) 90.7 (cid:139) 8.4 (n (cid:136) 3) ND 72.3 (cid:139) 9.1 (n (cid:136) 3) 70.0 (cid:139) 11.2 (n (cid:136) 3)

MiaPacaII POL-S RPA-S RPA-O HEK293 POL-S RPA-S RPA-O 66.8 (cid:139) 4.6 (n (cid:136) 4) 93.3 (cid:139) 7.6 (n (cid:136) 3) NA ND 68.3 (cid:139) 6.9 (n (cid:136) 3) 108.3 (cid:139) 1.7 (n (cid:136) 3) ND 69.0 (cid:139) 7.3 (n (cid:136) 3) 72.7 (cid:139) 8.2 (n (cid:136) 3) 15PC3

also effective, although less than the PS-version and only when assayed at 4 h, compatible with the intracellular instability of PO-ODN compared to PS-ODN. For Mia- PacaII cells, only the PS-ODN is effective, and the delivery method makes a big difference. HEK293 is a poor responder, although the anti-RPA70 PS-ODN performs better in electroporation than in liposomal transfection of these cells. The delivery by electroporation is more prone to variation, because most cells are killed by the shock, and only the surviving cells are assayed that are attached to the culture plastic at time of analysis. This yields a larger deviation than the liposomal delivery, where cells are attached to the growth surface from start to finish.

The cell internal fate of the ODNs was assayed with fluorescently labeled PS-ODNs using both delivery systems. With both methods, at least 90% transfection efficiency was obtained, and the cells displayed little variation in staining lines showed a similar uptake and intensity. All cell distribution, as shown in Fig. 4 (the nucleus was identified

treatment using liposomal transfection with 800 nM (i.e. 800 pmol) PS-ODNs directed against RPA70 (replication protein A, 70-kDa subunit), oncogene Ha-ras, and POL R2A (RNA polymerase II, 220 kDa subunit) on the respective target mRNA levels. Figure 3B shows the result using electroporation of 800 nM of antisense ODN directed against RPA70. A PS- as well as a PO-version of the ODN was used in those experiments. As PO-ODNs are quickly degraded by cellular nucleases, mRNA was assayed at 4 and 20 h post-transfection. The anti-RPA70 PS-ODN yields maximum efficacy already within 4 h post-transfection with liposomal delivery, at the same level as at 20 h post- transfection (A. L. M. A. ten Asbroek unpublished results). A summary of the quantification of the intact target mRNA levels is presented in Table 1. With liposomal delivery, the 15PC3 and MiaPacaII cells are the best responders, whereas HEK293 hardly responds at all. In 15PC3 cells, the anti- RPA70 PS-ODN displays the same potency with electro- poration as with liposomal transfection. The PO-ODN is

POL-S RPA-S RPA-O 19.3 (cid:139) 1.2 (n (cid:136) 3) 21.3 (cid:139) 0.5 (n (cid:136) 3) NA ND 28.0 (cid:139) 6.4 (n (cid:136) 3) 86.0 (cid:139) 9.3 (n (cid:136) 3) ND 35.0 (cid:139) 7.3 (n (cid:136) 3) 46.7 (cid:139) 4.1 (n (cid:136) 3)

Fig. 4. Staining pattern of cells 20 h after liposomal or electroporation transfection of FITC-labeled ODNs. HEK293 cells are much smaller than MiaPacaII and 15PC3, and therefore presented at an increased magnification.

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3H-labeled ODN uptake by cells (d.p.m.Ælg protein)1)

Table 2. 3H-labeled ODN (RPA-S) uptake of cells. 3H present in cells at 20 h post-transfection of a concentration series of antisense RPA-S is given as mean (cid:139) SD for two independent experiments. ND, not determined.

Liposomal Electroporation Sample

4.4 (cid:139) 0.0 6.4 (cid:139) 0.8 10.4 (cid:139) 0.9 7.8 (cid:139) 2.0 ND 24.2 (cid:139) 1.0

4.2 (cid:139) 0.6 6.8 (cid:139) 0.9 8.7 (cid:139) 0.4 ND ND ND MiaPacaII 400 nM 600 nM 800 nM HEK293 400 nM 600 nM 800 nM 15PC3

by Hoechst staining of the DNA; not shown). Liposomal transfection results mostly in a bright nuclear fluorescence that is excluded from the nucleoli and appears as bright spherical structures in a diffuse nucleoplasmic staining, as well as some cytoplasmic staining in bright punctate struc- tures. The electroporation transfection provides a completely different pattern, without detectable nuclear fluorescence, and with fine punctate perinuclear and cytoplasmic staining of other structures than appear following liposomal trans- fection. The corresponding PO-ODN shows a similar pattern and intensity as the PS-ODN in the fluorescent electropo- ration transfection (not shown). A tritium-labeled PS-ODN (against RPA70) was used in both delivery systems to quantify the amount of ODN that is retained in the cells at time of mRNA analysis. The amount of ODN per cell was quantified as 3H d.p.m. per lg protein and is shown in Table 2. The three cell lines assayed display similar cellular uptake. Thus, not only the intracellular distribution is similar for these cells (fluorescence), but also the intracellular concentration (tritium). Furthermore, the intracellular ODN concentration is a linear function of the ODN concentration administered at transfection (Table 2). Elec- troporation results in a roughly twofold higher concentration than liposomal delivery. Overall only 2–3% of the 3H-labeled ODN that is put into the transfection is still detected at 20 h post-transfection. The relative amount of tritium detected immediately after liposomal transfection is twofold higher for MiaPacaII and 15PC3 and fourfold higher for HEK293 compared to the 20 h data. This can largely be explained by cell division (as can be calculated from the total amount of protein measured at both time points).

The data obtained so far show that HEK293 cells have the lowest level of RNase H2 mRNA and display a very poor response to antisense ODN treatment. To test whether additional RNase H2 leads to enhanced sensitivity to ODNs, we constitutively expressed RNase H2 in HEK293

cells. Clones expressing high levels of RNase H2 RNA were assayed in vitro and in vivo. The in vitro RNase H assay, using whole cell extracts of the transfectants, shows that the expressed RNase H2 RNA yields functional protein, whereas the vector alone (panel pcV) does not affect the RNase H activity of the cells (Fig. 5A). The cell extracts of the RNase H2 transfectants (panels pcRH), have increased enzymatic activity. The lowest input (0.5 lL extract) already yields saturated enzyme activity levels. Activity could only be properly assayed using 10-fold diluted extracts (Fig. 5B). The cells overexpressing RNase H2 are (cid:25) 10-fold more active in this in vitro assay than the parental and vector control cells.

3.1 (cid:139) 0.3 6.5 (cid:139) 0.3 9.1 (cid:139) 1.4 5.4 (cid:139) 0.6 ND 13.4 (cid:139) 0.1 400 nM 600 nM 800 nM

Fig. 5. In vitro RNase H assay with whole cell extracts of HEK293 transfectant cells. (A)The parental HEK293 cells (293 wt) and typical examples of a pcDNA3 vector-only control transfectant cell line (pcV) and a pcDNA3/ RNase H2 transfectant cell line (pcRH8) using fresh extracts. (B) A vector-only control (pcV) and three pcDNA3/RNase H2 transfectants (pcRH8, pcRH9, pcRH10) that showed the highest level of RNase H2 RNA on Northern blot analysis, using 10-fold diluted extracts. In comparison with Fig. 5A, a lower level of digestion is obtained in all cases, because fro- zen extracts were used for the dilution, and freezing the extract leads to loss of activity in our hands (M. van Groenigen & A. L. M. A. ten Asbroek, published observations). However, the relative di(cid:128)erences in activity between the vector-only and RNase H2 transfectants are still retrieved.

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RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 589

alone showed a uniform distribution of fluorescence within the cells for all cell lines. The expression of the GFP–RNase H1 protein results in fluorescence throughout the whole cell in all cases, although the expression in 15PC3 seems to be less uniform. The expression of RNase H2 is restricted to only the nucleus (identified by Hoechst staining; not shown) in all cases except 15PC3. In these cells RNase H2 displayed the same uniform expression pattern as RNase H1.

Fig. 6. Northern blot analysis of 800 nM PS-ODN transfections of HEK293 cells overexpressing RNase H2. Cell lines shown are MiaPacaII (MPII), HEK293 (293), pcDNA3 vector-only control transfectant of HEK293 (pcV), RNase H2 transfectant cell lines of HEK293 overexpressing RNase H2 (pcRH8, pcRH9, pcRH10). PS-ODNs used are indicated on top of the lanes. aPOL, L5Cas20 directed against POLR2A; aRPA, ISIS12790 directed against RPA70; 20mer, randomized control mixture. Probes (indica- ted on the left) are for POLR2A (top), RPA70 (middle) or 28S rRNA (bottom).

D I S C U S S I O N

The RNase H2 overexpressing clones were tested in vivo using liposomal delivery of 800 nM PS-ODNs, directed against POLR2A and RPA70 (Fig. 6). Assaying the RNase H2 transfectants using electroporation was not feasible due to extremely poor plating efficiency of the RNase H2 overexpressing lines following electroporation, even on poly(L-lysine)-coated plates. All six RNase H2 transfectants assayed (three of which are shown in Fig. 6) had the same low level of antisense inhibition as the parental and vector control cells ((cid:25) 10% reduction of target mRNA). The high level of activity in vitro, and thus expression of functional protein, does not result in an increased response to antisense ODN treatment in vivo.

To rule out the possibility that different alleles of RNase H2 are expressed in MiaPacaII, HEK293 and 15PC3, we sequenced the coding region in these cells. The coding regions were identical, except for one silent substitution of the wobble base of a triplet encoding a proline residue. Position 579 (GenBank accession no. AF039652) is an A in MiaPacaII and 15PC3, but a G in HEK293.

In this study, we showed that the reduction of target mRNA upon treatment with ODNs against the 220 kDa subunit of RNA polymerase II, the 70 kDa subunit of replication protein A, and the oncogene Harvey-ras varies considerably between human cell lines. As the catalytic activity of an RNase H is essential for antisense-mediated RNA degra- dation we measured both mRNA and enzymatic activity. Large differences were observed in our cell lines in mRNA level of the two human RNase H enzymes. We focused on the comparison of the cell lines that displayed the major differences (Table 3). 15PC3 contains high levels of both RNases H1 and H2, MiaPacaII contains a low level of RNase H1 and a high level of RNase H2, whereas HEK293 contains a high level of RNase H1 and a low level of RNase H2 (10-fold more and fivefold less, respectively, than MiaPacaII cells as assayed by Northern analysis of total RNA). Despite these large differences in mRNA levels, we

The different response to antisense ODN treatment could also be attributed to a difference in enzyme localization within the various cell lines. To test this possibility, the coding sequences of RNase H1 and RNase H2 were fused in frame to green-fluorescent protein (GFP). The six cell lines were analyzed by fluorescence microscopy following tran- sient transfections (MiaPacaII, Hek293 and 15PC3 are shown in Fig. 7). Control experiments using the GFP vector

Fig. 7. Staining pattern of cells expressing green-fluorescent protein (GFP) and GFP fused to RNase H1 (GFP–H1) or RNase H2 (GFP–H2).

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Table 3. Summary of the results related to the involvement of RNase H1 and H2. For target mRNA reduction in vivo, +, 10–30% reduction of target mRNA level; + + +, 70–90% reduction of target mRNA level. ND, not determined; RH, RNase H.

Target mRNA reduction in vivo RH2 mRNA level

1.2 kb 5.5 kb Liposomal Electroporation RH1 mRNA level Cell line RH1 localization RH2 localization RH activity in vitro

+ +

As the fate of the ODNs within the different cell types was similar with respect to ODN accumulation and localization, a variation in response to ODN treatment must be an intrinsic property of the cells.

The mRNA data suggest that RNase H1 does not make a major contribution to the mRNA reduction of antisense treatment. Firstly, the three cell lines have similar RNase H in vitro activity, despite a big difference in RNase H1 mRNA levels, even when extracts are mixed. Secondly, the high level of RNase H1 in vivo in HEK293 compared to MiaPacaII does not result in an increased response to antisense ODN treatment, irrespective of the cellular ODN localization (liposomal delivery or electroporation of the ODNs). Finally, a GFP-RNase H1 fusion protein shows similar localization in all cell lines. This argues against a cell- specific restriction of RNase H1 to certain cellular com- partments. Rather it suggests that RNase H1, which is the ortholog of the minor E. coli enzyme RNase HII, with unknown function, is not a major player in the cell’s response to antisense ODN mediated cleavage of target mRNA.

detected a similar RNase H activity with the various cells when we used whole cell extracts in an in vitro RNase H assay. Single extracts displayed the same level of activity as mixed extracts, indicating that similar enzymatic activities were measured in the various extracts. In vivo, however, the cell lines showed a different response with a number of target mRNAs, which depended, in part, upon the delivery method used (Fig. 3). 15PC3 cells performed well for all three targets, yielding on average 80% reduction of the target mRNA, whereas HEK293 always performed poorly (only 20–30% reduction was achieved). The response of MiaPaca II cells depended on the ODN delivery method, yielding 70–80% reduction of the target mRNA with liposomal delivery and only 20–30% with electroporation. The amount of cellular ODN, measured with 3H-labeled PS-ODN, was twice as large after electroporation than after liposome-mediated transfection. FITC-labeling disclosed a large difference in ODN localization, which depended on the method of transfection. In our study, liposomal delivery of fluorescently labeled PS-ODNs resulted in a staining pattern that has been previously observed in various cell types, using different liposomes [37,38], or microinjection of PS-ODNs into the cytoplasm [38–41]. This pattern was independent of the ODN sequence, length, or the fluoro- chrome used [38,40]. The perinuclear and vesicular cyto- plasmic staining resulted from accumulation of ODN in the endosomes and lysosomes [37,41]. The bright nuclear ODN foci are the so-called PS-bodies, associated with the nuclear matrix; following mitosis they assemble de novo from diffuse PS-ODN pools in the daughter nuclei [38]. While they retain their antisense capacity, PS-ODNs continuously shuttle between the nucleus and the cytoplasm [42]. This nucleo- cytoplasmic shuttling is an active transport process, which probably involves binding to (unidentified) cellular proteins that exhibit shuttling. The nuclear localization of PS-ODNs seems to be an important prerequisite for their potential to exert antisense activity, despite their binding to nuclear matrix proteins [38].

The presence of two mRNA species, as well as a variation in the cellular localization complicates the interpretation of the role of RNase H2 (Table 3). The main 1.2-kb mRNA level varies substantially between the cell lines. In the in vitro RNase H assay, however, the three cell lines show similar cleavage activity. Thus, the activity measured in the in vitro assay does not correlate with the mRNA levels of either RNase H1 or H2. The discrepancy between the in vivo and in vitro measurements could be due to a compartmentaliza- tion of a component in the in vivo system. On the other hand, we cannot exclude the possibility that the substantial amount of 5.5-kb mRNA present in all cells encodes a major contributor of the RNase H activity measured in vitro. There are several examples of apparent discrepancies between RNase H activity measurements in different assays in mammals and yeast [36,43]. In mammalian cells the class I enzyme activity could only be measured in a liquid assay and was not detected with an in-gel assay; the class II activity measured in the liquid assay was of a monomeric enzyme, whereas the class II activity detected in-gel presented a multimeric enzyme form. In Saccharomyces cerevisiae, the class I activity was detected only in in-gel assays, the class II activity of RNH(35) only in liquid assays, whereas the class II activity of RNH(70) was detected in neither assay.

In order to determine the contribution of the activity encoded by the 1.2-kb RNase H2 mRNA, we assayed six

The pattern of ODN localization after delivery with electroporation was completely different, displaying no fluorescence at all in the nucleus. The cytoplasmic structures had a different appearance than those following the liposomal delivery; there were many more and they had finer punctate structures. After electroporation, the staining patterns observed with PO-ODNs and PS-ODNs are similar. This makes it unlikely that backbone chemistry- related binding components are involved in the cytoplasmic delivery of ODNs by electroporation.

+ + + + + + + + + + + +/– + + + + + + + + + + Whole Cell Whole cell Whole cell Whole cell Nucleus Whole cell Nucleus Nucleus + + + + + + + + + + + + + + + + + + + ND MiaPacaII + 15PC3 HEK293 HEK293 pcRH

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RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 591

15PC3 cells. This enzymatic location appears to be a necessity for activity towards ODNs that are restricted to the cytoplasm. On the other hand, the 5.5-kb mRNA species, whose sequence is unknown, awaits identification and characterization. We cannot exclude that it contributes to the activity essential for the antisense ODN-mediated inhibition of gene expression in vivo. This would be compatible with the finding that antisense ODNs can be very effective in inhibiting gene expression in the brain [44– 46]. In both fetal and adult brain, the main 1.2-kb RNase H2 mRNA can not (or hardly at all) be detected by Northern analysis (A. L. M. A. ten Asbroek, unpublished data; [20]), whereas they do have a consistent amount of the 5.5 kb RNase H2 mRNA species.

Our findings are not compatible with a simple assignment of a single RNase H enzyme activity to the antisense ODN- mediated inhibition of gene expression in human cells in vivo.

A C K N O W L E D G E M E N T S

We thank Dr K. Fluiter for performing the tritium labeling of the ODN, Prof. J. M. B. V. de Jong for critical reading of the manuscript, and our colleagues for helpful discussion and comments.

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