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In Vitro Transcription and Translation Inhibition by Anti-Promyelocytic
Leukemia (PML)/Retinoic Acid Receptor a and Anti-PML
Peptide Nucleic Acid
By Carlo Gambacorti-Passerini, Luca Mologni, Carla Bertazzoli, Philipp le Coutre, Edoardo Marchesi,
Franco Grignani, and Peter E. Nielsen
Peptide nucleic acids (PNAs) complementary to the15 bases
around thefusion point of both genomic DNA and cDNA of
the promyelocytic leukemia/retinoic acid receptor (Y (PMLI
RARa; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown bygel retardation
experiments t o specifically bind oligonucleotides corresponding t o t h efusion region of the P/R molecule. PNA was
also able t o successfully compete with anti-P/R DNA for duplex formation with P/R DNA and t o displace the anti-P/R
DNA from dsDNA. In vitro transcribed P/R RNA from two
inserts of ~ 3 5 and
0 -700 bp were testedin gel acceleration
experiments with fluorescein-conjugated PNA and showed
stable binding (resistant t o denaturing conditions) of PNA
to the newly transcribed RNA. Control RNA or transcripts
from thenoncoding strand did not bindPNA. However, this
PNA, although able t o specifically clamp polymerase chain
reaction, was incapable of inhibiting in vitro translation of
the PML/RARa mRNA, even when a bis-PNA was used.
Therefore, a PNA was targeted against the start region of
the P/R cDNA and against poly-purine regions of the gene.
Specific inhibition of in vitro translation and transcription
was shown, starting atconcentrations as low as 100nmol/L.
When oligonucleotides presenting the same sequence were
compared, PNA proved t o be approximately 40 times more
active. In conclusion, in vitro inhibition of translation and
transcription of the P/R gene can be obtained with PNA;
however, it is still necessary t o target the ATG start region
or poly-purine regions of the gene.
0 1996 by The American Society of Hematology.
T
95%. PNAs were synthesized asde~cribed.'~
Fluorescein-conjugated
PNA was obtained by reacting the PNA with fluorescein isothiocyanate. PNA sequences are as follows: no. 137 (complementary to the
genomic P/R fusion of patient no. 8), H-Gly-TCTCAAATGCTCCCT-NH2; no. 205, H-CTCAATGGCTGCCTC-NH2, and no. 496,
H-AATGGCTGCCTCCCC(eg1),(J),TJJ-NH2
(bis-PNA,"inwhich
J stands for pseudoisocytosine), complementary to the BCRl-type
fusioncDNA;no.752(complementarytothePMLstartcodon
no. 783(compleregion), H-CATGGTGGGCTCCTG-Lys-NH2;
mentary to the poly-purine region 1490-99 of the PML cDNA), HLys3-TJTTJTJJTI'(egl),TTCCTCTTCT-Lys-NH2;
and no. 753 (correspondingtothe
poly pyrimidineregion1433-40ofthePML
which is used
cDNA), H-Lys-JJTJTTJJ(egl),CCTTCTCC-Lys-NH,,
for in vitro transcription experiments.
1 in pGEM3).
PlasmidsBCRl(full-length
P/R cDNA,type
LAAB, and MOL (containing partial
P R inserts of 738 and 326
bases, respectively) were obtained fromP.G. Pelicci (Perugia, Italy).
Plasmid pCF-I (containing the full-length ret/PTC-2 cDNA") was
kindly supplied by I. Bongarzone (Milan, Italy).
PNA/DNA hybridization assay (gel retardation experiments).
Ten picomoles of an ODN (previously '2P-labeled at the 5'-end by
HE PROMYELOCYTIC leukemiahetinoic acid receptor a (PML/RARa; P R ) gene is present in virtually
all cases of acute promyelocytic leukemia (APL) and
derives
from a t( 15; 17) reciprocal chromosomal translocation' in
which part of the RARa gene on chromosome 17 is fused
to part of the PML gene on chromosome 15,' resulting in
the expression of a chimeric PMLIRARa (P/R) messenger
encoding a fusion P/R protein3
Growing evidence has suggested that the product of the
P/R gene is involved inboth the leukemogenesis process
and in the sensitivity of APL cells to retinoic acid (RA'.')
although the relative contribution to transformation of the
PML- and RARa-induced pathways hasnot yet been established.'.'
Peptide nucleicacid(PNA)'is
a DNA mimic inwhich
the (deoxy)ribose-phosphate backbone typical of DNA and
RNA is replaced by a charge-neutral, achiral pseudopeptide
backbone, preservingonlythebasespresent
in theDNA
molecule. PNA forms stable complexeswith complementary
sequences present in both dsDNA andRNA.'." In fact PNA/
DNA hybrids show an increase in Tm over corresponding
DNA/DNA complexes ofatleast
l"C/base.* Furthermore,
homopyrimidine PNAs bindveryefficiently and sequence
specifically tohomopurinesequences
in double-stranded
DNA by strand displacement. Finally, it has been found that
PNA can efficiently inhibit both translation and transcription
in vitro. Because of these properties, combined with a high
biologic stability, PNA holds promise as both an antisense
and an antigene drug. However, mostly model systems have
been studied"." and no general rules or conclusionsregarding the use of PNA for targeting biologically relevant genes
or sequences are yet available.
We present here resultswithseveral
PNAs directed
against the fusion region or the PML part of the P/R gene
and corresponding to both genomic and cDNA sequencesof
the BCR 1 -type fusion.3
MATERIALS AND METHODS
Oligonucleotides (ODNs), PNAs, and plasmids. ODNs were obtained from MedProbe SA (Oslo, Norway) at a minimum purity of
Blood, Vol 88, No 4 (August 15), 1996: pp 1411-1417
From the Division of Experimental Oncology D, lstitutoNazionale
Tumori, Milan, Italy: the Division of Internal Medicin III, Liidiwig
Maximilians University, Munich, Germany; the Department of Medicine, University of Perugia, Perugia, Italy: and the Center for Biomolecular Recognition, Biochemistry B, The Panum Institute, Copenhagen, Denmark.
Submitted November 28, 199s; accepted April 8, 1996.
Supported in part by the Italian Association for Cancer Research
(AIRC), the Italian Research Council (CNR No. 95.00842.CT04),
BIOMED-2 Grant No. BMH4-CT96-0848, CEVA, and the Danish
National Research Foundation.
Address reprint requests to Carlo Gambacorti, MD, Division of
Experimental Oncology D, Istituto Nazionale Tumori, Via Venezian
l , 20133 Milano, Italy.
The publication costs of this article were defrayed in partby page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8804-0035$3.00/0
1411
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1412
T4 polynucleotide kinase) were incubated for 30 minutes at
room
temperature (step 1) in TE buffer and then (step 2) at either room
temperature or 37°C for an additional30 minutes alone or with either
thecomplementaryODNorPNA.Thesamplesare
then run on
visualized by autoradiography. A
a 15% polyacrylamidegeland
competition assay was accomplished by incuhating in the same tube
both DNA and PNA. To verify the strand displacement ability of
PNA. P/R ODNs were incubated with their antisense ODNs during
step 1 (30 minutesat room temperature).Subsequently, PNA was
added and samples incubated for 30 additional minutes (step
2) at
either room temperature or 37°C.
PNA binding to P/R RNA (gelac~~leration
experinzenrs). Fluorescein-conjugated PNA (500 to I .000 ng) was incubated in TE buffer
with coldODN ( 1 to I O pg). with the transcriptionproducts(obtained from three reactions [see below]) from the LAAB and MOL
plasmid5 or with control RNA for30 minutes at 37°C. Samples were
then loaded on a 15% (ODNs) or 4% (transcripts) polyacrylamidegel
and run at 200 V. Gels were then mounted on 3M paper (Whntaman.
Maidstone. UK) anddried,andbandswerevisualized(asgreen
light) by direct UV illumination. Size markers were obtained by the
migration of RNA standards (RNA ladder; GIBCO. Gaithersburg.
MD) and detected on the wet gel by ethydium bromide staining and
UV transillumination. In someexperiments.sampleswere
mixed
one-third with formamide and loaded on 10 mol/L urea gels.
/ n v i f w transcription e.rprirnmts. Plasmids were first linearized
by digestion with Hind111 (used for MOL and anti-LAAB)o r EcoRI
(used for LAAB and anti-MOL). Template DNA was purified by
phenol/chlorofor-m extraction and used for in vitro transcription at
I pg/reaction (SP6iT7 transcription kit: Promega, Madison, W1)in
the presence of 20 pCi of [cU-’*P]UTP for I O to 30 minutes at 37”C,
as described.” For gel acceleration experiments, cold UTP wasused.
Transcription was initiated from the SP6 (LAAB and anti-MOL) or
T7 (MOL and anti-LAAB) site.
PNA was incubated with purified
DNA at 37°C for 30 minutes before starting transcription. UnincorporatedUTP was removed with NUC-TRAP mini columns(Stratagene. La Jolla CA), and the samples were run on 4% to 5 8 polyacrylamide gel electrophoresis(PAGE).dried.and
visualized by
autoradiography. The intensity of different bands was compared USing the 620 CCD densitometer (Bio-Rad, Richmond, CA) and the
l -D Analyst I1 data analysis software.
In rirro trunslrrfion experiments. Plasmids BCR I or pGF- 1 were
directly translated into polypeptides using
the TNT T7-coupled reticulocyte lysate system (Promega). Briefly. 1 pg DNA, T7 RNA polymerase,TNTbuffer,aminoacidmixture.
L-[ ”S]-methionine (20
pCi), RNasin (20 U; Promega). TNT rabbit reticulocyte lysate. and
PNA were mixed and incubated at 37 “C for 30 minutes, according
to TNT protocol. As internal controls, 0.5 pg of the pGF-I plasmid
was incubated in thesametuhe.Samples
were runin 6% to 7%.
sodium dodecyl sulfate-PAGE (SDS-PAGE), dried, and visualized
by autoradiography. Molecular weight markers (Bio-Rad) were run
in parallel.
Polymerase chain reaction (PCR) clamping. Anti-P/R PNA no.
205 and the bis-PNA no, 496 were used to target the fusion point
of P R in the BCRl plasmid. Two plasmids carrying the 8-glucocerebrosidase gene and the factor V gene were used as specificity controls.lx.l”Thefollowingprimerswere
used foramplification: 5’GGA GGC AGC CAT TGA GAC-3’ (forward) and 5’-CAT AGT
GGT AGC CTG AGG ACT T-3’ (reverse) for P/R, S’-GAA TGT
CCC AAG CCT TTG A-3‘ (forward) and 5‘-AAG CTGAAG CAA
GAG AAT CC-3’ (reverse) for P-glucocerebrosidase, and 5’-GGA
ACAACACCATGATCAGAGCA-3’(forward)andS’-TAG
CCA GGA GAC CTA ACA TGT TC-3’ (reverse) for factor
V. The
131, 357,and287
bp in
amplified fragmentswere,respectively,
length. PCR was performed in a 50 pL reaction containing 3 ng of
the plasmid, 200 pmol/L of each dNTP, 0.5 pmollL of each primer.
GAMBACORTI-PASSERINI ET AL
huffer (50 mmol/L KCI, 1 0 mmol/L, Tris-HCI, pH 8.3), 2.6 mmol/
L MgCI:. and 1.25 U Taq polymerase on the GenAmp PCR System
9600 machine (Perkin Elmer, Branchhurg,NJ) using a 4-stcpcycle’”:
88°C for 15 seconds. 62°C for l minute. 54°C for 20 seconds. 72°C
for 90 seconds for 25 cycles. PNA was either preincuhated overnight
with the plasmid o r added i n the tube at the beginning of thc reaction.
RESULTS
Anti-P/R PNA is able to hind specijically to P/R DNA and
to successfully compete with anti-P/R DNA (gel retardation
experiments). To investigate the ability of PNA to bind P/
R DNA, 15 mer ODNs corresponding to the fusion region
of the P/R gene were synthesized and used in gel retardation
experiments.
Two types of P/R fusions were studied. Initially. the sequence corresponding to genomic fusion (from the patient
identified as no. 8) was targeted. In this patient. the fusion
region (AGAGTTTACGAGGGA) has an approximate A+G
content of 75%. Gel retardation experiments are shown in
Fig 1A. Both anti-P/R DNA and PNA can bind to P/R DNA.
Anti-P/RDNA slows the migration of P/R DNA (lanes 2
and 3. consistent with the formation of dimers). PNA forms
two bands (lanes 4 and 5 ) that migrate more slowly than the
DNA/DNA dimers because of the lack of net charges on the
PNA molecule. Thesetwobandsarecompatible
with the
formation of a PNNDNA duplex (lowerband) and of a
nonperfect PNAJDNA triplex. As expected, the triplex predominates when working in an excess of PNA (lane 4). If
DNADNA dimers are allowed to form and then are challenged with PNA(lane 61, then about 48% of the DNA/
DNA complexis displaced, with the formation of PNNDNA
dimers (and triplexes, upper band). However, if the second
incubation is performed at 37°C instead of at room temperature, then a near complete displacement of the DNADNA
dimers is observed(Fig IB, lanes X through 1 I), showing
the superior binding efficiency of PNA. Anti-P/R PNA does
of S and7
not bind to PML or RARa ODNs (mismatch
bases. respectively) or to anti-P/R ODN. PNA also doesnot
interfere with the binding of PML (Fig IA, lanes 9 through
1 1 ) and RARa (data not shown) ODNs to their complementary ODNs.
Subsequently,ODNscorrespondingto
the BCRl type
cDNA (GAGGCAGCCATTGAG) wereused (Fig IC). This
represents a mixed sequence with only 66% A+G content:
thus, only PNNDNA duplexes are observed (lanes S through
7). PNA successfully competed with DNA(lane 8, 77%
PNA/DNA I: 23% DNA/DNA). Anti-P/R PNA is ableto
successfully displace anti-P/R DNA from DNADNA dimers
and to bind specifically t o P/R ODN when incubated with
the DNA/DNA dimers forat least 60 minutes at 37°C (lanes
I O through 12).
These experiments show that PNA is able to specifically
bind to ODN representing both the genomic and the cDNA
P/R fusion region and to successfully compete with anti?/
R DNA for binding to P/R DNA.
Ability of’ PNA to hind transcripts qf the P/R gemJ (gel
accelercrtionexperiments).
P/R RNAwas transcribed in
vitro from the two plasmids LAAB and MOL that contain
~ 7 0 0 and
c350-bp inserts of the P R cDNA, including
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1413
PNAINHIBITS PMVRARa GENE TRANSLATION
A
1
2
3
4
5
6
1
2
3
4
5
6
7 8 9
6
7
8
7 8 9
10
11
-
PNAz/DNA
PNA/DNA
-
DNAIDNA
-
SSDNA
Fig l. Ability of anti-P/R PNA to bindspecifically
t o both single-stranded DNA (ssDNA) and doublestranded DNA IdsDNA) representing fusion regions
containing poly-purineor mixed sequences. (A) A 15
mer ODN representing a genomic P/R fusion region
(AGAGmACGAGGGA, high purine 175961 content)
PNA2/DNA
named P/R was labeled and used. Lane 1, P/R alone
(10pmol); lanes 2 and 3,P/R + anti-P/R ODN (100
and 10 pmol, respectively); lanes 4 and 5,P/R + antiPNAlDNA
P/R PNA (100and 10 pmol, respectively); lane 6, P/
R
anti-P/R ODN (100pmol, step 1) followed by
PNA 1100 pmol, step 2 [room temperature]); lanes 7
DNAlDNA
and 8, P/R
anti-PML or anti-RARe ODN; lane 9,
labeled PML ODNalone; lane 10, PML
anti-PML
ODN; and lane 11, PML anti-PML ODN anti-P/R
PNA. (B) Lane 1, P/R alone 110 pmol); lanes 2 through
4,P/R anti-P/R ODN (100.10,and 5 pmol, respecSSDNA
tively); lanes 5 through 7, P/R + anti-PIR PNA 1100.
10, and 5 pmol, respectively); lanes 8 and 9, P/R
anti-P/R ODN (10pmol, step 1)followed by anti-P/R
PNA (step 2, 37°C; 100 and 10 pmol, respectively);
lanes 10 and 11, same as lanes 8 and 9 but containing
anti-P/R ODN at 5 pmol/L. (C)Ability ofanti-P/R PNA
t o bind an ODN representing the fusion region
of the
P/R cDNA, a non-poly-purine DNA sequence. A 15
mer ODN representing the BCR1-type fusion region
(GAGGCAGCCATTGAG, mixed sequence), named
004, was labeled and used.Lane 1, 004 alone 110
pmoll; lanes 2 through 7, 004 + anti-PIR ODNs (10, I'NAIDNA
30, and 100 pmol, lanes 2 through 4,respectively) or
PNA (lanes 5 through 7); lane 8,004 anti-P/R ODN
and PNA; lanes 9 through 12, 004 anti-P/R ODN DNNDNA(100pmol, step 1) followed by PNA (100pmol, step
2 [37"Cll for 30 minutes llane 9) or for 60, 120, or
SSDNA
240 minutes (lanes 10 through 12, respectively). All
samples were run in 12% PAGE. The bands corresponding to ssDNA, dsDNA, PNA/DNA, and PNAJ
DNA are indicated.
10
11
-
-
+
+
+
+
+
+
-
+
C
1
2
3
4
5
9 1 0 1 1 1 2
+
+
-
the fusion point. Because PNA does not contain phosphate
molecules, conventional binding techniques could notbe
used. Therefore, a fluorescein-conjugated PNA-based technique was used.PNAisnot
charged and therefore is expected to have little migration when electric current is applied. The binding to RNA would confer mobility to PNA,
whichcan be visualized as a green signal by direct UV
illumination of the dried gel. An example using ODNs is
presented in Fig 2A. PNA no. 205 (500 ng [=l00 pmol]),
which binds specifically to P R ODN, can migrate into the
gel and forms a discrete band. Down to 40 pmol of PNA
can be visualized (data not shown). Transcripts from the
MOL and LAAB plasmids (containing partial P R inserts of
326 and 738 bases, respectively) were then incubated with
PNA and run on PAGE (Fig 2B through D). Unbound PNAs
show little shift, as expected. When incubated with P R transcripts, PNA migrates (gel acceleration) at positions compatible with the length of the transcripts. Incubation ofPNA
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GAMBACORTI-PASSERINI ET AL
1414
A
B
1
2
3
4
2
5
3
4
5
l
1.6,
1.0-
0.6PNNODN
0.4-
-
0.3-
C
l
2
3
4
D
5
1
2
3
1.6-
1.0-
0.6-
-
1 .oPNA
PNAAWA
0.4 -
0.3-
- PNA
Oa6-
0.4-
- PNA/RNA
0.3-
Fig 2. Ability of anti-P/R fluorescein-conjugated PNA t o bind t o P/R mRNA transcribed by plasmids LAAB (738bases) and MOL(326bases)
or t o P/R ODNs. (A) PNA (100pmol) was incubated for
30 minutes at room temperature with
anti-P/R ODN (lanes l and 2,100 and 1,000 pmol.
respectively) or with anti-PML or anti-RARa ODNs (lanes 3 and 4, 1,000 pmol). Lane 5, PNA alone. Samples were run on1596 PAGE. PNA was
visualized as described under the Materials andMethods. (B) PNA (lane 5)was incubated for 30 minutes at 37°C with transcripts from MOL
(lane l), LAAB (lane 2). or their antisense transcript (lanes 3 and 4). (C) PNA (lane 5)was incubated (as in [B]) with control RNA (lanes 1 and
2, 10
and 20 pg, respectively) and with transcripts from LAAB (sense [lane 31 and antisense [lane 41). (D) Lane 1, PNA alone; lane 2, PNA +
anti-MOL RNA; lane 3, PNA + MOL RNA. Samples were mixed with formamide
and run in denaturing (10mol/L urea) PAGE. Molecular markers
are indicated.
with antisense transcripts or with unrelated control RNAs
did not induce any shift of the PNA (Fig 2C, lanes 1 and
2). Whereas control PNAs enter thegel in Fig 2C, they
apparently fail to form a band in Fig 2B. Although a clear
explanation is not available, it is possible that, in the experiment depicted in Fig 2B, a partial precipitation of PNA
occurred that prevented control samples from forming a distinct band.
The binding of PNA to its target is very stable, because,
once formed, it is resistant to denaturing ( I O molL urea)
conditions (Fig 2D). In conclusion, PNA is able to bind
specifically P/R RNA and forms a stable complex.
PNA-mediated inhibition of PCR amplification of P/R.
To establish a possible functional role for the observed binding of PNA no. 205 (and of its corresponding bis PNA no.
496) to nucleic acids, we performed a PCR clamping test?"
In this assay, the specific arrest of Taq polymerase-mediated
amplification of a P/R sequence by PNA is assessed. PNA
no. 205 failed to affect the amplification of a 13 l-bp fragment containing the P/R fusion point, whereas PNA no. 496
showed significant ( 250%) inhibition in the pmolL range.
The block was significantly increased when PNA no. 496
was preincubated overnight with template DNA, reaching a
significant inhibition at 300 nmol/L and a total block at 2
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1415
PNA INHIBITS PMURARa GENE TRANSLATION
Fig 3. Inhibition of PIR polypeptide synthesis by
PNA no. 752 directed against the PML start region.
The upper band ( 4 1 0 kD) refersto theP/R products,
whereas the lower band (76 kD) refersto the product
of pGF-l (internal control). PNA concentrationswere
0, 50, 100, 300, and 500 nmolIL (lanes 1 through 5,
respectively).
116-
1
2
W
W&
3
4
a P/R protein
m
8 0 - II) @:
pmol/L (data not shown). PCR inhibition by PNA no. 496
was sequence-specific, because parallel amplifications of pglucocerebrosidase and factor V gene fragments werenot
affected (data not shown).
In vitro translation experiments. The ability of anti-P/R
PNA to inhibit the in vitro translation of the P/R fusion
protein from the expression plasmid BCRl was evaluated.
PNA no. 205, whichshowed a stable binding to the P/R
fusion region, was added to the in vitro translation assay,
but failed to inhibit the translation of the P/R polypeptide
from plasmid BCRl (data not shown). A second PNA (no.
496), synthesized as a bis-PNA" against the fusion region,
inhibited translation at high (>2 pmol/L) concentrations, but
in a nonspecific way (data not shown). Similarly, both PNAs
failed to inhibit the transcription of the noncoding strand of
MOL and LAAB.
Because the targeting of the fusion region proved unfeasible, PNAs were directed against the PML start codon region
(no. 752, residues 122-136) and against a poly-purine region
(no. 783. residues 1490-99) of PML, whichisretained in
the fusion gene, to inhibit translation.
Figure 3 shows the results using PNA 752 to inhibit in
vitro translation of the P/R proteinfromplasmid
BCRI.
Specific inhibition starts at 50 to 100 nmol/L and complete
inhibition is achieved at 300 nmol/L. Translation of the control plasmidpGF-I (lower band) was unaffected. The activity
of PNA 752 was also compared with an ODN with the same
sequence (Fig 4A and B). The ODN achieved a significant
translation inhibition at 8 pmol/L. The concentrations of
200 nmol/L (PNA) and 8 pmol/L (ODN) achieved similar
(=80%) translation inhibition. The activity of PNA 783 was
also evaluated (Fig 4C). At 300 nmol/L, a 60% inhibition
was obtained (compared with 100% for PNA 752); no accumulation of a truncated product was evident. Further increases in concentration led to nonspecific inhibition.
In vitro transcription experiments. A bis PNA (no. 753)
targeted against a homopurine stretch in the PML gene was
used to inhibit in vitro transcription from the LAAB plasmid
(Fig 5 ) . This PNA would be expected to block transcription
approximately 100 bases after the T7 promoter site. A truncated =lOO-base-Iong transcript became evident at 500
nmol/L, progressively increased in intensity at 1 and 2 pmol/
L, and reached 30% of the total transcript (as assessed by
densitometric analysis). A proportional decrease of the fulllength transcript was also observed. Further increases in
PNA concentration resulted in the nonspecific inhibition of
both the full-length and the truncated products.
These experiments show that PNAs directed against the
5
U
d
Control
protein
start codon or poly-purine regions canspecificallyinhibit
both in vitro transcription and translation of the P/R gene.
PNA can achieve a degree of translation inhibition similar
to that obtained using 40 times higher concentrations of
ODN. Inhibition of transcription canbeachieved by targeting a poly-purine region in the coding DNA strand.
A
1
2
3
6
5
6
7
8
9
116-
a P R protein
n\
loo
concentration [ n w
1
116-
2
"-
lu;*
3
4
5
6
7
a P/R protein
Q
Control protein
Fig 4. Comparison between PNAno. 752 and a samesequence
ODN or a different PNA on translation inhibition. (AI Same legend
as Fig3. Lane 1, control translation; lanes2 and 3,100 and 200nmoll
L PNA, respectively; lanes4 through 9,200 nmol/L, 400 nmol/L, 800
nmol/L, 2 pmolIL, 4 pmol/L, and 8 pmol/L ODN, respectively. (B)
Densitometric analysis from data presented in Fig 3 and (A): 0,
PNA
752; M, ODN. Protein levels were calculatedasareas;nonspecific
inhibition (as evidenced by the decrease in pGF-1 bands [eg, in lane
81) was subtracted. (C) Lane 1, control; lanes 2 through 4, 100, 300,
and 600 nrnol/L PNA 752, respectively; lanes 5 through 7,300, 600,
and 900 nrnol/L PNA no. 783, respectively.
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GAMBACORTI-PASSERINI ET AL
1416
800 500 400
full length
msCriPt
-
300 -
160 truncated
mnscript
Fig 5. Inhibition of in vitro transcription from LAAB plasmid by
PNA no. 753. Lane 1, control transcription; lanes 2 through 4, 6, and
7, preincubationwith PNA at 0.5, 1,2,4, and 8 pmollL, respectively;
lane 5, empty. The position of molecular markers is indicated.
DISCUSSION
The hybrid protein P/Repitomizes one of the growing list
of fusion molecules generated by chromosomal translocations associated with human cancer. The causal role of the
P/R protein and of most other fusion proteins in the pathogenesis of human cancer is now well established, although
the molecular mechanism by which each protein transforms
cells is still obscure in many cases. Our ability to modulate
the expression andor the function of fusion proteins is even
more limited at present.
PNA represents a promising tool in this direction. PNA
could potentially be used as both an antisense and an antigene molecule. We initially verified the DNA-binding ability
of PNA using labeled ODN representing the fusion region
of the P/R molecule. Subsequent experiments confirmed that
anti-P/R PNA could bind to transcripts from the P/R gene.
Unfortunately, even a stable binding to the fusion region
of the mRNA was notsufficient to inhibit P/R protein synthesis, although specific termination of PCR amplification of a
fragment containing the P/R fusion was attained. It is possible that the ribosomes could displace the bound PNA and
continue synthesis downstream. These data also show the
difficulties present when trying to precisely target the fusion
region of hybrid genes, mainly due to the limited variability
and number of sequences that can be generated. However,
this problemisnot restricted to the targeting of DNA or
RNA, but is also present when different strategies (eg, the
elicitation of an immune response against the fusion region
of the P/R
are followed.
Subsequently, the potential target region was extended to
the entire PML part of the fusion molecule. Because it is
knownthatnon-RNase
H-activating ODN analogues can
have better translation inhibition when targeted to the initiation codon, a new15-merPNA
was prepared. With this
PNA, a specific and sensitive translation inhibition was obtained at concentrations as low as 100 nmol/L. This repre-
sents the first description of translation inhibition obtained
by PNA-mediated targeting of a mixed (non-poly-purine)
sequence corresponding to a naturally occuring gene. Translation inhibition by PNA has been shown so far only when
targeting homopurine sequences13 artificially introduced in
nonbiologically relevant genes.
When the same sequence ODN was evaluated, it appeared
to be several times less effective than PNA on a molar basis.
A different PNA (no. 783), directed against a homopurine
region of the cDNA, also exhibited some activity at 300
nmol/L; however, this became nonspecific at higherconcentrations. This observation requires further investigation. It is
important to note that, when specific translation inhibition
was obtained withPNAno. 783, no accumulation of a truncated product was found, which is at variance with in vitro
transcription (see below). This fact could be explained with
the successive block of several translation complexes at different positions and the ensuing release of polypeptides of
distinct molecular weight that, therefore, do not form a single
band. The absence, or nonstoichiometrical amount, of a truncated translation product has been observed in other systems
too.23
A bis-PNA directed against a homopurine region of the
coding strand was used to evaluate transcription inhibition.
In this case, a progressive accumulation of a truncated product (with decrease in the full-length transcript) was observed,
although athighPNA concentrations, close to the micromoles per liter mark, and without reaching a complete suppression. In this case, higher concentrations produced nonspecific inhibition of boththe full-length and truncated
transcript.
Although additional important information are notyet
available, including the ability of PNA to enter cells:4 these
data support the possible use of PNA as a specific tool to
modulate gene expression. Although results inintact cells
are an essential prerequisite for clinical development, these
data show that an oncogene canbetargetedatboththe
translation and transcription level and epitomize potentials
and limitations of targeting different regions of the gene. In
this context, it is worth saying that, although the targeting
of the PML start (ATG) regionwouldnotbeabsolutely
tumor-specific, the function of PML in normal cells could
be easily vicariated, as data in PML knock-out mice seem
to suggest (P.P. Pandolfi, unpublished observations).
The observed inhibition of transcription, although not
complete, is particularly promising, because it is known that
only few molecules (2 in theory) need to be blocked inside
a cell to completely inhibit transcription. PNA could thus be
used as an antigene molecule, with the aim of selectively
blocking gene transcription.
ACKNOWLEDGMENT
The authors thank Drs G. Parmiani for discussion, G. Manenti
for helpful criticism and support, and J. Barrett and N. Young for
reviewing the manuscript.
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1996 88: 1411-1417
In vitro transcription and translation inhibition by anti-promyelocytic
leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide
nucleic acid
C Gambacorti-Passerini, L Mologni, C Bertazzoli, P le Coutre, E Marchesi, F Grignani and PE
Nielsen
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