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Experimental Therapeutics |
and PML Expression in Acute Promyelocytic Leukemia Cells by Anti-PML Peptide Nucleic Acid1
Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy [L. M., E. M., C. G-P.], and Center for Biomolecular Recognition, IMBG, The Panum Institute, DK2200N Copenhagen, Denmark [P. E. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is tightly linked to the pathogenesis of acute promyelocytic leukemia (APL); hence, it represents a tumor-associated, transformation-related molecule. In this study, three anti-PML adamantyl-conjugated peptide nucleic acid (PNA) oligomers previously described as in vitro inhibitors of PML/RAR translation were combined and used to block PML/RAR synthesis in NB4 cells. Cationic liposomes were used to achieve sufficient delivery of PNAs into the cells. Upon treatment of cells with the liposome/PNA mixture, enhanced cellular uptake of PNA (approximately 5-fold compared with control) was obtained. Concomitantly, a substantial reduction (>90%) of the expression of PML/RAR was observed when all of the three PNAs were used together. This resulted in a dramatic effect on the number and viability of NB4 cells in culture after 48 h of treatment. This phenomenon was preceded by induction of apoptosis that could be observed 24 h after treatment. No sign of granulocytic differentiation was observed after treatment. These effects were also noted on other leukemic cell lines that express PML but not the fusion transcript. These results show that it is possible to deliver PNA into hematopoietic cells and obtain specific gene inhibition, and they suggest that a growth inhibitory effect on acute promyelocytic leukemia cells can be obtained through the block of PML/RAR and PML expression. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene, yielding the chimeric gene PML/RAR (reviewed in Ref. 1
). Although APL is not a very common disease (10% of all of the acute myeloid leukemias), it has the peculiarity of being the first example of cancer that can be treated with a specifically targeted differentiation therapy. Indeed, it is known that RA can induce complete (although temporary) remission of the disease, and its use in combination with chemotherapy has dramatically increased the cure rate for this disease (2)
. Interestingly, both the leukemogenesis and the high response to RA are linked to the presence of the PML/RAR fusion protein.
The PML/RAR OFP is causally linked to the development of APL (3)
; therefore, it represents an optimal target for treatment strategies aimed at obtaining a selective inhibition of the growth of APL cells through the down-regulation of the gene product responsible for the leukemogenesis. Results obtained by targeting another OFP, Bcr/Abl (4)
, support such a strategy.
Antisense oligodeoxynucleotides represent a unique example of gene-specific drugs that can be used to selectively inhibit the expression of target genes (5) . Second and third generation oligodeoxynucleotides have been synthesized that possess improved chemical characteristics regarding stability in biological fluids, cellular uptake, and molecular specificity for the target sequence (6) . Among the various alterations of the standard phosphodiester structure that have been conceived, the PNA backbone (7) is optimal in terms of specificity and affinity for the target and resistance to degradation. PNA is a structural mimic of natural nucleic acids, composed of a pseudo peptide carrying nucleobases. PNA/DNA hybrids are more stable than double-stranded DNA (8) . PNA is resistant to degradation caused by nucleases and proteases, and it has been shown to interfere in a sequence-specific manner with several DNA- and RNA-based processes (9 , 10) . One major concern relating to the therapeutic use of PNA is its poor ability to enter the cells. Thus far, few studies (11, 12, 13, 14, 15) have successfully addressed this issue, showing intracellular effects of PNA.
To develop a gene-specific treatment of APL, we studied previously (16
, 17)
the potential of PNAs as in vitro blockers of transcription and translation of the PML/RAR gene in cell-free assays. A PNA targeted against the internal AUG translation initiation codon of PML/RAR could block the synthesis of the protein driven from that site (16)
. A combination of three different PNAs was required to completely inhibit the translation from the complete mRNA, containing the first AUG and the 5'-untranslated region (17)
. In the present study, we used these three PNAs to inhibit the expression of PML/RAR in an APL-derived cell line, NB4, that carries the t(15;17) rearrangement (18)
. The PNAs were chemically modified by the covalent addition of the lipophilic adamantyl group to allow interaction of the oligomers with liposomes (19)
. We show here that such Ada-PNAs can be effectively delivered into NB4 cells by means of liposomes and that treatment of NB4 cells with liposomes containing the three anti-PML PNAs induces significant reduction in PML/RAR protein cellular levels and growth inhibition attributable to cell death. These data suggest the possible use of liposomes as carriers of PNA through the cell membrane and encourage the development of PNA as an effective tool in tumor-specific therapy. This study also shows that inhibition of PML and PML/RAR expression in NB4 cells is followed by development of apoptosis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PNA#1 [Ada-Fl-GCAGGCTCCATGGAC-LysNH2, where Ada represents an adamantyl group (19)
, Fl stands for fluorescein, and T is a lysine backbone-modified thymine unit (21)
] is directed against the PML/RAR first AUG start codon (17)
. PNA#2 [Ada-Fl-CATGGTGGGCTCCTG-LysNH2] is targeted toward the second AUG. PNA#3 [Ada-Fl-AGATCTTGGAGTGCG-LysNH2] hybridizes to a sequence in the 5'-untranslated region of the transcript. PNA#4 [Ada-Fl-AGTTCTAGAGGTGCG-LysNH2] is a scrambled version of PNA#3 in which four nucleobases have been interchanged, so that it contains four mismatches relative to PNA#3. It was designed as a sequence-specificity control PNA. All of the PNAs were obtained by solid-phase synthesis as described (21, 22)
, dissolved in DMSO, aliquoted, and stored at -20°C until needed.
Cell Treatment and Analysis.
DMRIE-C reagent (Life Technologies, Inc.) was used to introduce PNAs into the cells in a standard transfection protocol as suggested by the manufacturer.
Cells were harvested after 18 h of treatment, washed extensively in PBS, and analyzed for PNA uptake by flow cytometry (FACScan; Becton Dickinson) to detect cell-associated fluorescence. An aliquot of cells from each sample was used to prepare cytospins (700 rpm for 4 min) or dropped on a glass slide and viewed under a fluorescence microscope to determine cellular localization of fluorescent PNA.
Cells were harvested and counted after 2 days of treatment for analysis of PNA effects. Total cell lysate corresponding to 2 x 105 cells was run on a 7.5% polyacrylamide gel and probed with rabbit anti-RAR (C-20; Santa Cruz Biotechnology), goat antiactin (I-19; Santa Cruz Biotechnology) polyclonal antibodies, or monoclonal anti-PML antibody (PG-M3; Ref. 23
). Intensity of bands was analyzed by the Eagle Eye II video system and the Eagle Sight 3.2 software (Stratagene). Immunofluorescence was performed on cytospins as described in Topcu et al. (23)
.
Quantitative determination of viable, early and late apoptotic cells was carried out by FACS analysis using the annexin V/propidium iodide-binding technique, as described previously (4) . Samples were washed in PBS and kept for 15 min in an annexin V/propidium iodide mixture before the analysis at the FACScan. Compensation for double fluorescence was performed by staining the samples either with annexin V or with propidium iodide only.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and microscopy (Fig. 2)
analysis showed fluorescence associated with the cells. A PNA concentration-dependent fluorescent signal was detected by the flow cytometer (Fig. 1, A–C)
when the cells were treated with the PNA:liposome preparation. Samples treated with PNA alone showed very little fluorescence, as shown by Fig. 1D
. Note that PNA used at the concentration of 1 µM with liposomes (Fig. 1C)
shows a stronger signal when compared with PNA alone at 5 µM, suggesting that liposomes induced a more than 5-fold enhancement of PNA uptake into NB4 cells.
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to confirm that fluorescence measured by FACS was derived from live cells rather than aggregates of precipitated material. Fig. 2B
clearly indicates the homogeneous presence of PNA inside living cells after incubation with PNA-loaded liposomes. Finally, we wanted to check that PNA was actually penetrating the cells and not just associated to the cell surface and to study the intracellular localization of PNA. To this end, we analyzed cytospins from each sample: PNA diffused throughout the cytoplasm, with less evident nuclear staining (Fig. 2C)
. These experiments indicate that effective delivery of Ada-PNAs to NB4 cells can be obtained using liposomes as carriers.
Antisense Activity of Anti-PML PNAs in APL Cells.
Cells were treated for 48 h with either empty liposomes or the three anti-PML PNAs alone, or with liposomes containing antisense PNAs or the control PNA. Total lysates were then prepared for Western blot analysis. NB4 cells treated with liposomes carrying a mixture of PNA#1, PNA#2, and PNA#3 showed a marked reduction of PML/RAR expression (Fig. 3)
, whereas normal RAR was not affected (data not shown). The extent of the inhibition depended both on the concentration of the lipids (Fig. 3A
; compare Lane 3, 65% inhibition as assessed by densitometric analysis, with Lane 4, 85% inhibition) and on the concentration of the three PNAs. The inhibitory effect increased as the concentration of each PNA increased from 1 µM (Lane 8, no substantial inhibition) to 5 µM (Lane 9, 60% inhibited) and 10 µM (Lane 4, 85%). Almost total inhibition (>90%) was observed when each of the three PNAs was present in the cell culture at the concentration of 12 µM (Fig. 3B)
. Individually, each of the three PNAs showed no significant inhibition. The negative control for sequence specificity consisted of a scrambled version of PNA#3 used at a concentration equal to the sum of the concentrations of the three antisense PNAs (Fig. 3C)
. It caused some reduction in PML/RAR protein (30%), possibly attributable to non-antisense effects. However, no effect on the growth and viability of the cells was evident (see below). By contrast, inhibition of 85% (Fig. 3C)
and 90% (Fig. 3B)
was obtained by the PML-specific PNAs used at the same total concentration. PML/RAR protein levels were always normalized over the intensity of the corresponding actin band to eliminate experimental variation in gel loading. Because our PNAs are directed against PML sequences, nonrearranged PML was also evaluated (Fig. 3D)
. PNA-treated cells showed decreased expression of PML, whereas no inhibition was caused by empty liposomes or control PNA#4.
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protein expression can be obtained in NB4 cells by the use of antisense PNAs targeted to the regions that control the translation of the two transcripts.
Biological Effect of Antisense Treatment.
After treatment and before collection of the samples for Western blot assay, cell count and viability were determined by trypan blue exclusion assay. The data in Fig. 4
report the number of living cells at the end of treatment (compared with the untreated control) and represent an average of three experiments. The number of viable NB4 cells (Fig. 4A)
was significantly reduced above 8 µM of each PNA (with a calculated IC50 between 8 and 10 µM) at concentrations at which PML/RAR expression was also remarkably affected, indicating a correlation between PML/RAR protein intracellular levels and cell growth. The cell viability of the antisense-treated samples (evaluated as the percentage of cells that excluded the dye) decreased with a similar curve (data not shown), suggesting that the reduced number of cells was attributable to cell death rather than to growth arrest. Treatment of NB4 cells with equal amounts of the control PNA#4 did not affect the number of cells (Fig. 4A)
or the viability (data not shown). To discriminate between the effect of PML/RAR synthesis inhibition from that attributable to PML down-regulation, we treated PML/RAR-negative myeloid leukemic cells LAMA and HL-60 that express normal PML with the three anti-PML PNAs (Fig. 4B)
. The treatment caused a similar level of growth inhibition and cell death, suggesting that the observed phenotype was mediated by a decrease of PML protein. Empty liposomes, scrambled PNA, or PNA#1 used alone at 24 µM did not cause inhibition (Fig. 4B)
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. Treatment with liposomes and PNAs #1, #2, and #3 (12 µM each for 24 h) induced apoptosis in 20% of the cells whereas 4% of control cells (treated with liposomes carrying scrambled PNAs) were found in early apoptotic state.
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or nitroblue tetrazolium reduction (data not shown). Cells surviving the PNA treatment were recultured in 10-6
M RA and were found to be responsive as control cells. Immunofluorescence analysis with an anti-PML antibody (23)
showed no apparent rearrangement of PML oncogenic domains but rather a decreased signal, in line with antisense down-regulation of PML protein (data not shown).
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in hematopoietic cells. This in turn resulted in decreased cell growth and apoptosis, while no apparent differentiation was induced. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antisense PNAs (16, 17)
. PNA was chosen because of its superior binding properties and higher stability in biological media, as compared with DNA-based or RNA-based antisense molecules like oligodeoxyribonucleotides and ribozymes. These features and a relatively easy synthesis and vast possibility of chemical modification make PNA a very attractive molecule. In this study, we investigated the possibility of suppressing PML/RAR expression in NB4 cells, using three antisense PNAs specific for the 5'-untranslated region and the two AUG codons present in PML/RAR. These PNAs were shown previously (17)
to cause complete and specific inhibition of PML/RAR translation in a cell-free system only when used in combination. Also, in another case (25)
we observed that optimal translation inhibition by PNA required targeting of multiple sequences, even in the presence of a single AUG codon. Unfortunately, cells take up naked PNA very poorly, and efficient cellular delivery systems are needed to exploit its formidable antisense potential. Here, we report on the use of liposomes as carriers of anti-PML/RAR Ada-PNAs into NB4 cells. Liposomes were able to increase significantly the cellular uptake of these PNAs. The amount of free PNA that could enter the cells was negligible, whereas a clear and diffuse mostly cytoplasmic localization was observed when PNA was delivered by liposomes. Diffusion of PNA into the nucleus of the cells was also observed in some instances, but it was limited to a minor proportion of cells (data not shown). These data demonstrate that liposomes provide an efficient delivery of Ada-PNA into NB4 cells.
The consequences of the introduction of the anti-PML/RAR PNAs in NB4 cells are described in Figs. 3
4
5
. PML/RAR protein synthesis was strongly and specifically inhibited, and the cells showed a dramatic reduction in number and viability and developed apoptosis. The fact that neither PNA alone nor lipids alone caused any inhibition indicates that these effects were attributable to the action of PNA and not to unspecific toxicity of the treatment. Furthermore, this effect was not observed when a scrambled PNA (#4) was used. Therefore, the inhibition of NB4 cell growth is believed to be caused by a specific antisense activity of PNA. These results support the view that the efficacy of PNA as an antisense molecule observed in cell-free systems can be extended to the cellular level, emphasizing a potential use of PNAs for the control of gene expression. Our data represent the first evidence that PNA can specifically affect gene expression in intact hematopoietic cells, although they also show that this effect can be achieved only when an intracellular delivery system, such as liposomes, is coupled to the administration of PNA. However, relatively high concentrations of PNA (10 µM) were needed in these experiments, compared with the in vitro translation system where substantial inhibition was achieved at PNA concentrations between 0.1 and 1 µM. Thus, although it was technically possible to deliver PNA into living cells at biologically relevant concentrations, more efficient carriers are required to increase the in vivo efficacy of this approach. Different alternative methods for introducing PNAs into eukaryotic cells are currently under development (15
, 26, 27)
.
Treatment of NB4 cells with the PNAs did not lead to differentiation, in agreement with the observations of Nason-Burchenal et al. (28, 29)
, who described the triggering of apoptosis in the absence of granulocytic maturation in NB4 cells transfected with an anti-PML/RAR ribozyme. After treatment, the remaining cells were still able to differentiate in the presence of RA; however, this is difficult to interpret, because most cells were dying at the end of the experiment and the few cells that survived long enough to respond to RA might be those ones that did not internalize PNA. These cells could not be assayed for PML/RAR expression, because they represented a minority in PNA-treated populations. In fact, in these populations, PML/RAR levels correlated with the number of live cells. RA-treated cells could not be tested for expression at the end of RA treatment either, because RA induces PML/RAR degradation per se (1)
. However, because PML/RAR is required for NB4 cells to respond to RA (1
, 30)
, it is likely that these cells do express the fusion protein.
It is well established that a substantial reduction of PML/RAR expression in NB4 cells can affect cell growth and viability. In our case, however, a similar effect was obtained in leukemic cells that do not express PML/RAR, indicating that suppression of normal PML may also be responsible for the apoptotic response of NB4 cells that we observed. PML is thought to exert a pro-apoptotic action in cooperation with Daxx (31)
, and PML-deficient mice show impaired apoptosis (32)
. However, a null phenotype in knockout mice does not necessarily have the same biological effect of an acute inhibition of expression in cells expressing the gene. Moreover, Daxx null embryos die of extensive apoptosis (33)
, and PML is overexpressed in papillary thyroid carcinoma (34)
and other tumors. Therefore, the picture might be more complicated than what is currently known, and the effects of PML down-modulation could be different in various cell types and different experimental conditions.
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FOOTNOTES |
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1 Supported in part by the Italian Association for Cancer Research (AIRC, 420.198.662); the Italian Research Council, Istituto Superiore di Sanità (881A/10); the Italian Ministry of Health; BIOMED 2 Grant BMH4-CT96-0848; and EU-TMR Grant BMH4-CT96-5006. 
2 To whom requests for reprints should be addressed, at Department of Experimental Oncology, Istituto Nazionale Tumori, via Venezian 1, 20133 Milan, Italy.
3 The abbreviations used are: APL, acute promyelocytic leukemia; PML, promyelocytic leukemia; RA, retinoic acid; RAR, RA receptor; OFP, oncogenic fusion protein; PNA, peptide nucleic acid; Ada-, adamantylated; FACS, fluorescence-activated cell sorter.
Received 4/24/00. Accepted 5/16/01.
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, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood, 93: 3167-3215, 1999. transgene initiates murine acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA, 94: 2551-2556, 1997. and anti-PML peptide nucleic acid. Blood, 88: 1411-1417, 1996. gene. Nucleic Acids Res., 26: 1934-1938, 1998. translocation product triggers apoptosis in promyelocytic leukemia cells. Oncogene, 17: 1759-1768, 1998.[Medline]
is lethal to retinoic acid-resistant promyelocytic leukemia cells. Blood, 92: 1758-1767, 1998. protein. Blood, 82: 1573-1577, 1993.This article has been cited by other articles:
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  T. Shiraishi, R. Hamzavi, and P. E. Nielsen Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells Nucleic Acids Res., August 1, 2008; 36(13): 4424 - 4432. [Abstract] [Full Text] [PDF]
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T. Shiraishi and P. E. Nielsen Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates Nucleic Acids Res., September 15, 2004; 32(16): 4893 - 4902. [Abstract] [Full Text] [PDF]
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N. Dias and C. A. Stein Antisense Oligonucleotides: Basic Concepts and Mechanisms Mol. Cancer Ther., March 1, 2002; 1(5): 347 - 355. [Full Text] [PDF]
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