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

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
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 t he cells was
liposome-mediated or via electroporation, which resulted
in dierent intracellular locations of the ODNs. The
ODN-mediated target mRNA reduction varied consider-
ably between the cell lines. In view o f 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 e xamined in this study.
The i ntracellular localization of the enzymes, assayed by
green-¯uorescent protein fusions, showed that RNase H1
was present throughout the whole cell for all cell types
analyzed, whereas RNase H2 w as restricted to the nucleus
in all cells except the prostate cancer line 15PC3 that
expressed the protein throughout the c ell. Whole cell
extracts of the cell lines yielded similar RNase H cleavage
activity in an in vitro liquid assay, in contrast to the
ecacy of the ODNs in vivo. Overexpression of RNase
H2 did not aect the r esponse t o O DNs in vivo. Our data
imply t hat in vivo RNase H activity is not only due to the
activity assayed in vitro, but also to an intrinsic p roperty
of the cells. RNase H1 is not likely to be a major player
in the a ntisense ODN-mediated degrada tion o f t arget
mRNAs. RNase H2 is involved in the activity assayed
in vitro. The presence of cell-type speci®c factors aecting

the activity and localization of RNase H2 is strongly
suggested.
Keywords: ribonuclease; RNase H ; human; antisense;
phosphorothioate.
Ribonucleases H (RNases H) are enzymes that speci®cally
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 f unctionally understood RNases H are the
RNase H domains of retroviral reverse transcriptases, and
the evolutionary related RNase HI of Escherichia coli.For
both t hese enzymes, the c rystal structures are available [4,5]
and amino-acid residues involved in substrate binding,
metal binding, and catalysis h ave been identi®ed and studied
in detail by site-directed mutagenesis [6,7]. Mammalian
RNase H enzyme activities have been biochemically c har-
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 h igher eukaryotes has be en
grouped into two classes [13,14]. Class I enzymes have a
native molecular mass of 68±90 kDa, are activated by both
Mg
2+
and Mn
2+
, and are active in the presence of
sulfhydryl reagents. C lass II e nzymes h ave a l ower molec-

ular mass (30±45 kDa), are activated only by Mg
2+
and
inhibited by additional Mn
2+
, and are highly sensitive to
sulfhydryl-blocking reagents.
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 identi®ed and characterized [17±21]. This
has helped t o link the biochemically characterized enzyme
activities to the gene sequences. An overview o f the two
RNase H families, and their homologues identi®ed 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 p rokaryotic 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-speci®c 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:
Abbreviations: ODN, oligodeoxynucleotide; PS, phosphorothioate;
PO, phosphodiester; RNase, ribonuclease; FITC, ¯uorescein; GFP,
green-¯uorescent protein.
(Received 13 July 2001, revised 16 November 2001, accepted
17 November 2001)
Eur. J. Biochem. 269, 583±592 (2002) Ó FEBS 2002
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 modi®ed 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-speci®c
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].
In our search for allele-speci®c inhibitors based on single-
nucleotide polymorphisms in target mRNA sequences using
antisense PS-ODNs, which could provide a tumor cell
speci®c 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 t arget sequences
and ODN delivery methods. Furthermore, w e investigated
the role of R Nase H2 in this process using in vitro and in vivo
measurements.
MATERIALS AND METHODS
Cell culture
Human cell lines HEK293 (embryonal kidney), 15PC3
(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 modi®ed Eagle's medium (DMEM), supple-
mented with 10% fetal bovine serum, 2 m
ML
-glutamine,
100 UámL
)1
penicillin, and 100 lgámL
)1
streptomycin.
Transfections
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 250 3
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  70% con¯uency was
reached. Cells were harvested using trypsine followed by
washing in NaCl/P
i
, and resuspended in Hepes-buffered
media (2 m
M
Hepes, 15 m
M
K-phosphate buffer, 250 m
M
mannitol, 1 m
M
MgCl
2
,pH7.2;[32])at10
6
cells per
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
complete culture medium and plated at appropriate density
for recovery.
Plasmid transfections for t ransient expression of GFP-
constructs were with 2 lg supercoiled plasmid on 10
5
cells.
For ¯uorescence 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 ®xed on the glass in NaCl/P
i
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 ®lters. For stable expression of
RNase H2 in HEK293, cells were plated in 10-cm dishes at
10
7
cells and transfected for 5 h in 2.5 mL serum-free
medium containing 12.5 lL transfe ction 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

Tritium l abeling of the ODN was performed using the heat
exchange method described b y Graham et al. [33]. Cells
were transfected with
3
H-labeled PS-ODN ( speci®c activity
40 260 d.p.m.álg
)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/P
i
(5 ´ 3mL NaCl/P
i
per well) and lysed sub-
sequently in 1 m L 1
M
NaOH p er w ell. Aliquots of 500 lL
were used fo r liquid scintillation counting. Prote in concen-
tration was measured with Bio-Rad DC r eagent (Bio-Rad)
using a BSA standard series for quanti®cation.
Plasmids
C-Terminal GFP fusion vector pEGFP-C1 was o btained
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 veri®ed by
DNA sequencing using Big-Dye terminator chemistry
(PerkinElmer) and analyzed on an ABI377 sequencer.
RNA analysis
Northern blot analysis of RNA w as carried out as
described previously [29]. Hybridized probe was visual-
ized and quanti®ed 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).
584 A. L. M. A. ten Asbroek et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In vitro
RNase 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: e xponentially growing cells were
harvested by scraping, washed once in NaCl/P
i
,and
resuspended in 100 lL hypotonic lysis buffer (7 m
M
Tris/
HCl p H 7 .5, 7 m

M
KCl, 1 m
M
MgCl
2
,1m
M
2-mercapto-
ethanol) per 10
6
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 m
M
Tris/
HClpH7.5,116m
M
KCl, 3.6 m
M
MgCl
2
,6m
M
2-mercaptoethanol) was added. Cell debris was removed
by centrifugation for 10 min at 4 °C. The supernatant w as
transferred to a fresh tube o n ice and glycerol was added to a
®nal 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 t he
extracts of different c ell lines has to be compared. Absolute
levels differ between the e xperiments. 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
for5 minin100 m
M
KCl, 0.1 m
M
EDTA and slowly cooled
to room temperature to allow folding of the template.
Template RNA (50 ng) and 100 n g ODN were annealed at
37 °Cfor15minin30lL 100 m
M
KCl, 0.1 m
M
EDTA.
Then, RNase H mixture was added, comprised of 8.4 lL
5 ´ buffer (250 m
M
Tris/HCl pH 7.5, 50 m
M
MgCl
2
,1m
M
dithiothreitol, 2.5 mgámL
)1

BSA), 1 lLRNasin(20UálL
)1
;
Promega) and 5 lL cell extract, and incubated at 37 °Cfor
5 m in. RNA was subsequently precipitated in the presence
of glycogen, after removal of proteins by phenol extraction,
and dissolved in gel loading buffer c ontaining 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.
RESULTS
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 M iaPacaII
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 t o a ntisense 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 1 5PC3
display a similar high level of RNase H1, whereas MiaP-
acaII, T24 a nd HTB82 show a low level. The difference in
intensity between the two groups, after normalization for
28S rRNA signal, is about 10-fold. The RNase H2 mRNA
level shows a ®vefold to 10-fold variation, but with a
different distribution over the cell lines. 15PC3 and Mia-
PacaII display the highest l evel 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]tobea
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 t o twofold). Our subsequent
analysis focused on the three cell lines that present the
possible variation in mRNA levels: M iaPacaII (low RNase
H1, high RNase H2), HEK293 (high RNase H1, low RNase
H2) and 15PC3 (high RNase H1, high RNase H2).
As mRNA levels do not n ecessarily re¯ect 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 i n a concentration series
to assay the nonsaturated part of the activity curve, and
mixtures of two different cell e xtracts were compared to the
Fig. 1. Northern blot analysis of RNases H in the cell lines. Total RNA
isolated from expone ntially growing cells was hybridized to prob es 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.
Ó FEBS 2002 RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 585
separate extracts. A representative example o f a n R Nase H
assay is shown in Fig. 2 (Fig. 2 A 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 R Nase H in the extracts. Roughly 10% of the
input RNA remains uncut. In both cases, the range f rom 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 d ifference in activity obtain ed w ith
the combined extracts i n Fig. 2 A,B re¯ects the interexp eri-
mental variation. The fact that the combined extracts are as
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). P hosphorothioate (PS) ODNs behave similarly to
PO-ODNs in the in vitro assay. They are slightly less
ef®cient, 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
ef®cacy of a particular ODN can therefore b est be a ddressed
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
Fig. 3. Northern blot analysis of the cell lines transfected with 800 n
M
ODNs directed against RPA70 and Ha-ras or POLR2A. Pro bes used
are i ndicated on the left side. 28S rRNA and GAPDH hybridization
were used for quanti®cation 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 m ixture of 20-mer PS-ODNs; mock, transfection without PS-
ODN. RNA w as iso lated for a nalysis a t 20 h post-transfection (B)

Electroporation transfection of 800 n
M
PS-ODN ISIS 12790 (RPA-S)
and the PO version of this O DN (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 w ith whole cell extracts of cell lines
HEK293 (A) and MiaPacaII (B). Theamountofextract(XT)usedis
indicated on top of the lanes. The lanes depicted 0.5 + 0.5 and 1 + 1
are ass ayed with a mixt ure of both cell e xt rac ts. D i gest ed pr o duct i s
detected as a single band on t hese gels, as the ODN h ybridizes 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 lane s as
percentage of total signal detected in the lane (remaining uncut input
RNA plus digested product RNA).
586 A. L. M. A. ten Asbroek et al. (Eur. J. Biochem. 269) Ó FEBS 2002
treatment using liposomal transfection with 800 n
M
(i.e.
800 pmol) PS-ODNs directed against RPA70 (replication
protein A, 70-kDa subunit), oncogene Ha-ras,andPOL
R2A (RNA polymerase II, 220 kDa subunit) on the
respective target m RNA levels. Figure 3B shows the result
using electroporation of 800 n
M
of antisense ODN directed
against RPA70.APS-aswellasaPO-versionoftheODN
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 ef®cacy 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 quanti®cation 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 a s with liposomal transfection. The PO-ODN is
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 t he 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 t hat 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 ®nish.
The cell internal fate of the ODNs was assayed with
¯uorescently labeled PS-ODNs using both delivery systems.
With both methods, at least 90% transfection ef®ciency was
obtained, and the cells displayed little variation in staining
intensity. All cell lines showed a similar uptake and
distribution, as shown in Fig. 4 (the nucleus w as identi®ed
Table 1. Percentage of intact target mRNA after antisense ODN treatment. After treatment with 800 n
M

antisense ODNs, phosph orothioate (POL -S
and RPA-S ) o r phosphodiester (RPA-O), cells were assayed f or intact target m RNA at 20 h or 4 h post-transfection, using Northern blotting.
Percentages, corrected for l oading and normalized to the mock control transfections, are given as mean  SD for n independent experiments. ND,
not determined; NA, not available, as PO-ODNs do n ot enter c ells when delivered by liposomal transfection reagents.
Sample
Delivery system
Liposomal 20 h Electroporation 20 h Electroporation 4 h
MiaPacaII
POL-S 19.7  3.3 (n  6) ND ND
RPA-S 26.0  2.2 (n  3) 80.7  9.0 (n  3) 72.3  9.1 (n  3)
RPA-O NA 90.7  8.4 (n  3) 70.0  11.2 (n  3)
HEK293
POL-S 66.8  4.6 (n  4) ND ND
RPA-S 93.3  7.6 (n  3) 68.3  6.9 (n  3) 69.0  7.3 (n  3)
RPA-O NA 108.3  1.7 (n  3) 72.7  8.2 (n  3)
15PC3
POL-S 19.3  1.2 (n  3) ND ND
RPA-S 21.3  0.5 (n  3) 28.0  6.4 (n  3) 35.0  7.3 (n  3)
RPA-O NA 86.0  9.3 (n  3) 46.7  4.1 (n  3)
Fig. 4. Staining pattern of cells 20 h after
liposomal or electroporation transfection of
FITC-labeled O DNs. HEK293 cells are
much smaller than MiaPacaII and 15PC3, and
therefore p resented at an increased
magni®cation.
Ó FEBS 2002 RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 587
by Hoechst staining of the DNA; not shown). Liposomal
transfection results mostly in a bright nuclear ¯uorescence
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 ¯uorescence,
and with ®ne punctate perinuclear and cytoplasmic s taining
of other structures than appear following liposomal trans-
fection. The corresponding PO-ODN shows a similar p attern
and intensity as the PS-ODN in the ¯uorescent electropo-
ration transfection (not shown). A tritium-labeled PS-ODN
(against RPA70) was used in both delivery systems to
quantify the amount of ODN t hat i s r etained in the cells at
time of mRNA analysis. The amount of ODN per cell was
quanti®ed as
3
H d.p.m. p er lgproteinandisshownin
Table 2 . The three cell lines assayed display similar ce llular
uptake. Thus, not only the intracellular distribution is similar
for these cells (¯uorescence), but also the intracellular
concentration (tritiu m). Furthermore, the intracellu lar
ODN concentration is a linear function of the ODN
concentration a dministered at transfection (Table 2). Elec -
troporation results in a roughly twofold higher concentration
than liposomal delivery. Overall only 2±3% of the
3
H-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 HE K293 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 c onstitutively expressed R Nase H2 in HEK293
cells. Clones expressing high levels of RNase H2 RNA were
assayed in vitro and in vivo.Thein vitro RNase H assay,
using whole cell extracts of the transfectants, shows that the
expressed RNase H2 RNA yields f unctional 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), h ave increased
enzymatic activity. The lowest input (0.5 lLextract)already
yields saturated enzyme activity levels. Activity could only
be properly assayed using 10-fold diluted extracts (Fig. 5B).
The cells overexpressing RNase H2 a re  10-fold more
active in this in vitro assay than the parental and vector
control cells.
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 H 2 transfectant cell line (pcRH8) using
fresh extracts. (B) A vector-only control (pcV)
and three pcDNA3/RNase H2 tran sfectants
(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 a ll cases, because fro-
zen extracts were used for the dilution, and
freezing the extract leads to loss o f activity in
our hands (M. vanGroenigen &A. L. M. A. ten
Asbroek, published observations). However,
the relative dierences in activity between the
vector-only and RNase H2 transfectants are
still retrieved.
Table 2.
3
H-labeled ODN (RPA-S) uptake of cells.
3
H present in cells
at 20 h post-transfection of a concentration series of antisense RPA-S
is given a s mean  SD for two independent experiments. ND, no t
determined.
Sample
3
H-labeled ODN uptake by cells
(d.p.m.álg protein
)1
)
Liposomal Electroporation
MiaPacaII
400 n
M
4.4  0.0 7.8  2.0
600 n
M

6.4  0.8 ND
800 n
M
10.4  0.9 24.2  1.0
HEK293
400 n
M
4.2  0.6 ND
600 n
M
6.8  0.9 ND
800 n
M
8.7  0.4 ND
15PC3
400 n
M
3.1  0.3 5.4  0.6
600 n
M
6.5  0.3 ND
800 n
M
9.1  1.4 13.4  0.1
588 A. L. M. A. ten Asbroek et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The RNase H2 overexpressing clones were tested in vivo
using liposomal delivery of 800 n
M
PS-ODNs, directed
against POLR2A and RPA70 (Fig. 6). Assaying the RNase

H2 transfectan ts using electroporation was not f easible d ue
to extremely poor plating ef®ciency of the RNase H2
overexpressing lines following electroporation, even on
poly(
L
-lysine)-coated plates. All six RNase H2 transfectants
assayed (three o f which are s hown in Fig. 6) had the s ame
low level of antisense inhibition as the parental and vector
control cells ( 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.
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-¯uorescent protein (GFP). The s ix cell lines
were analyzed by ¯uorescence microscopy following tran-
sient transfections (MiaPacaII, Hek293 and 15PC3 are
shown in Fig. 7 ). Control experiments using the GFP v ector
alone showed a uniform distribution o f ¯uorescence within
the cells for all cell lines. The expression of the GFP±RNase
H1 protein results in ¯uorescence throughout the whole cell

in all cases, although the exp ression in 15PC3 seems to be
less uniform. The expression of RNase H2 is r estricted to
only the nucleus (identi®ed by Hoechst staining; not shown)
in all cases except 15PC3. In these cells RNase H2 displayed
the same uniform expression pattern as RNase H1.
DISCUSSION
In this study, we showed that the reduction of target mRNA
upon treatment w ith 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 l ow 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 ®vefold less, respectively, than
MiaPacaII cells as assayed by Northern analysis of total
RNA). Despite these large differences in mRNA levels, w e
Fig. 6. Northern blot analysis of 800 n
M
PS-ODN transfections of HEK293 cells
overexpressing RNase H2. Cell lines s hown
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 th e lanes. aPOL,
L5Cas20 directed against POLR2A; aRPA,
ISIS12790 directed a gainst RPA70;20mer,
randomized control mixture. Probes (indica-
ted on the left) are for POLR2A (top), RPA70
(middle) or 28S rRNA (bottom).
Fig. 7. Staining pattern of cells expressing
green-¯uorescent protein (GFP) and GFP
fused to RNase H1 (GFP±H1) or R Nase H2
(GFP±H2).
Ó FEBS 2002 RNases H and variation in cellular response to ODN (Eur. J. Biochem. 269) 589
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 a s
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, meas ured with
3
H-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 ¯uorescently 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 ¯uoro-
chrome used [38,40]. The perinuclear and v esicular cyto-
plasmic staining resulted from accumulation of ODN in the
endosomes and lysosomes [37,41]. The b right nuclear ODN
foci are the so-called PS-bodies, associated with t he 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 cyto plasm [42]. This nucleo-
cytoplasmic shuttling is an a ctive transport p rocess, which
probably involves binding to (unidenti®ed) 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 pattern of ODN localization after delivery w ith
electroporation was completely different, displaying no
¯uorescence at all in the nucleus. The cytoplasmic structures
had a different appearance than those following the
liposomal delivery; there were m any m ore a nd they had
®ner punctate structures. After electroporation, the s taining
patterns observed with PO-ODNs and PS-ODNs are

similar. This makes it unlikely that b ackbone ch emistry-
related binding components are involved in the cytoplasmic
delivery of ODNs by electroporation.
As the fate of the ODNs within the dif ferent 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 activi ty, despite a big difference in RNase H1
mRNA levels, even w hen 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 o r electroporation of the
ODNs). Finally, a GFP-RNase H1 fusion protein shows
similar localization in all cell lines. This argues against a cell-
speci®c 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.
The presence of two mRNA species, as well as a variation
in the cellular l ocalization comp licates 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 s everal examples of apparent discrepancies
between RNase H activity measurements in di fferent assays
in mammals and yeast [36,43]. In mammalian cells the class
I enzyme activity c ould only be measured i n a liquid assay
and was not detected with an in-gel assay; the class II
activity measured in the liquid assay was o f a monomeric
enzyme, whereas the class II activity detected in-gel
presented a multimeric enzyme form. In Saccharomyces
cerevisiae, t he class I activity was detected only in in-gel
assays, the class II activity of RNH(35) only in liquid a ssays,
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 H 2 mRNA, we ass ayed six
Table 3 . Summary of th e results rela ted to the involvement of RN ase H1 an d H2. For target mRNA r eduction in vivo, +, 10±30% reduction of
target mRNA level; + + +, 70±90% reduction of target mRNA level. ND, not determined; RH, RNase H.
Cell line
RH1
mRNA
level
RH2
mRNA level
RH1

localization
RH2
localization
RH activity
in vitro
Target mRNA
reduction in vivo
1.2 kb 5.5 kb Liposomal Electroporation
MiaPacaII + + + + Whole Cell Nucleus + + + + +
15PC3 + + + + + + + Whole cell Whole cell + + + + + + +
HEK293 + + + +/± + Whole cell Nucleus + + +
HEK293
pcRH
+++ +++++ + Whole cell Nucleus + + + + ND
590 A. L. M. A. ten Asbroek et al. (Eur. J. Biochem. 269) Ó FEBS 2002
different transfectant clones of HEK293 (three of these are
showninFigs5and6)thatexpressedaspectrumofhigh
levels of RNase H2, up to a 25-fold higher level than the
endogenous 15PC3 RNase H2 mRNA. The increase in
RNase H2 RNA in the transfectants resulted in increased
enzymatic activity in the in vitro RNase H assay. This
demonstrates that the overexpressed R Nase H2 contributes
substantially to the enzymatic activity assayed in w hole c ell
extracts. However, these HEK293 transfectants overex-
pressing functional RNase H2 do not display an increased
response to antisense ODN treatment in vivo.Duetoan
increased fragility of the transfectants, it was not possible to
analyze the effects of ODNs delivered by electroporation.
The data of the 15PC3 cells are compatible with the
hypothesis that RNase H2 can play a role in the in vivo

response of c ells. They a re the only cells that show a good
response to antisense ODN treatment using electroporation
of PS- and PO-ODNs. With this transfection method the
ODNs (PS as well as PO) are only detected in the cytoplasm.
15PC3 cells are the only cells that have RNase H2 protein
both in the cytoplasm and the nucleus, as opposed to a strict
nuclear localization in the other ce ll lines tested. Thus the
cytoplasmic localization of RNase H2 in 15PC3 might be
responsible for the catalytic a ctivity after electroporation of
antisense ODNs. The cytoplasmic RNase H2 is not an
absolute requirement for effective antisense inhibition, as
MiaPacaII cells displaying nuclear ¯uorescence of GFP±
RNase H2 show a similar reduction of the target mRNA as
15PC3 cells when PS-ODNs are transfected with liposomes.
However, nuclear location of RNase H2 is not suf®cient
for ODN-mediated mRNA degradation. HEK293 and
MiaPacaII cells display a similar localization of RNase H2,
as well as similar ODN localization and accumulation.
Nevertheless, HEK293 cells do not respond to PS-ODN
treatment, even when they express vast amounts of active
enzyme.
Reviews discussing PS-ODN-mediated inhibition of gene
expression warn against erroneous interpretation of r esults
caused by the protein-binding capacity of PS-ODNs [27,28].
The lac k o f r eactivity o f H EK293 c ells in our study could
therefore simply be explained by postulating a c ell-speci®c
factor that inac tivates the PS-ODNs in these cells, which
would imply that this factor is inactive in the in vitro RNase
H assay, or that some other enzymatic activity is measured.
The detection of increased activity in the transfectants

overexpressing the coding region of the 1.2-kb RNase H2
mRNA suggest s that at le ast the ac tivity encoded b y the 1.2-
kb mRNA can be assayed in vitro. On the other hand, the
fact that 15PC3 cells display RNase H2 not strictly in
the nucleus as the other cells, but also in a large amount in
the cytoplasm, clearly shows that cell-speci®c components
exist t hat a ct o n this RNase H enzyme. As w e d educe t he
cellular localization from the behavior of the GFP-RNase
H2 fusion protein, the cellular factor must act with the
RNase H2 enzyme. The p reviously mentioned nucleocyto-
plasmic shuttling of PS-ODNs with the help of shuttling
cellular components [42] may play a role in the cell-speci®c
variation in response to antisense ODN treatment.
A clear assignment of the role of RNase H2 in the PS-
ODN mediated cleavage of t arget mRNA in vivo requires
some additional knowledge. On the one hand, the compo-
nents binding to this enzyme need to be identi®ed to
understand the cytoplasmic location of the enzyme in
15PC3 cells. This enzymatic location appears to be a
necessity for activity t owards ODNs that are restricted to
the cytoplasm. On the other hand, the 5.5-kb mRNA
species, whose sequence is unknown, awaits identi®cation
and c haracterization. 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 ®nding that antisense ODNs can be
very effective in inhibiting gene expression in the brain [44±
46]. In both fetal and adult b rain, t he 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 k b RNase H2 mRNA species.
Our ®ndings are not compatible with a simple assignment
of a single RNase H e nzyme activity to the antisense ODN-
mediated inhibition of gene expression in human cells
in vivo.
ACKNOWLEDGEMENTS
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 c omments.
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