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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 731-737
PLENARY PAPER
From the Department of Cell Growth and Differentiation, Institute of
Microbiology and Biochemistry, CSIC/University of Salamanca, Salamanca,
Spain.
One major obstacle to the effective treatment of cancer is to
distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are
ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as
a target model the abnormal BCR-ABL p190 and p210 products, we
constructed M1-RNA with guide sequences that recognized the oncogenic
messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the
effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we
studied in vitro and in vivo effects of these RNA enzymes against
BCR-ABLp190 and
BCR-ABLp210, bearing in mind that both
fusion genes share the ABL sequence but differ in the sequence
coming from the BCR gene. We showed that
M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the
guide sequence (GS). We also demonstrated that when M1-p190-GS and
M1-p210-GS were expressed in proper mammalian cell models, they
abolished the effect of BCR-ABL by specifically decreasing the
amount of the target BCR-ABL mRNA and preventing the function of the
BCR-ABL oncogenes. These data clearly demonstrate the
usefulness of the catalytic activity of M1-GS RNA to cleave
specifically the chimeric molecules created by chromosomal
abnormalities in human cancer and to represent a novel approach to
cancer treatment.
(Blood. 2000;95:731-737)
A key difficulty in the effective treatment of patients
with cancer is distinguishing between tumor cells and normal cells. This is why current treatments for cancer are often ineffective. There
have been remarkable advances in our understanding of the molecular
biology of cancer that suggest new mechanisms for the selective
destruction of tumors.1-3 The molecular characterization of
tumor-specific chromosomal abnormalities has revealed that fusion
proteins are involved in most types of cancer.4-6 These fusion proteins result from chimeric genes created by the translocation and production of chimeric mRNA species that contain exons from each
gene involved in the translocation. Chimeric molecules are ideal
therapeutic targets because they are unique to the disease; they only
exist in the tumor cells, not in the patient's normal cells.1-3 Inhibition of chimeric gene expression by
antitumor agents specifically kills leukemic cells without affecting
normal cells.
As therapeutic agents, zinc-finger proteins,3 antisense
RNA,1,2,7 or hammerhead-based ribozymes8,9 have
been used. All these methods have some limitations. Zinc-finger
proteins must act at the DNA level, interacting with the desired
sequence and blocking transcription. However, gene fusions at the DNA
level occur within introns, and this implies that a new zinc-finger has
to be designed for every patient. Thus, directing the strategy at the
mRNA level seems to be more practical. Antisense molecules, either
oligodeoxynucleotides or antisense RNA, act in a 1:1 stoichiometric relationship. This problem can be overcome with the use of ribozymes that, because of catalytic activity, process and destroy a higher number of target molecules per molecule of ribozyme. However, hammerhead ribozymes, in turn, require presence of specific nucleotide sequences in the target RNA to be cut, and these requirements cannot
always be fulfilled. These data imply that new therapeutic tools would
be desirable to allow the inactivation of any chimeric fusion gene product.
M1 RNA is the catalytic RNA subunit of RNase P from Escherichia
coli. RNase P is a ribonucleoprotein complex that catalyzes the
hydrolysis reaction that removes a 5' leader sequence from tRNA
precursors and several other small RNAs of similar structure. Studies
of substrate recognition by M1-RNA and RNase P10-12 have led to the development of a general strategy of gene targeting in which
M1 RNA can be used as a tool to cleave any specific mRNA sequence
simply by the 3' terminal addition to the ribozyme sequence of a
so-called guide sequence (M1-GS) complementary to the target mRNA, that
forms a base-pair with it and leaves a 5'-ACCAC-3' unpaired
stretch needed for the M1-GS RNA to recognize and cleave this
artificially created substrate13 (Figure
1C). Thus, M1-GS RNA, apart from some
requirements to improve its cleavage efficiency,14 can be
specifically directed to cut any mRNA sequences. We have taken
advantage of this property to destroy the tumor-specific fusion genes
created as a result of chromosomal translocations.
We have used as a model target, a well-characterized example in the
hematopoietic system that involves the rearrangement of the BCR
and ABL genes in Philadelphia chromosome-positive
(Ph1+) chronic myelogenous leukemia and acute lymphoblastic
leukemias. This translocation results in the formation of
chimeric BCR-ABL oncogenes.15-24 Depending of the
precise breakpoint within the BCR gene, fusion proteins of 210 kd (p210) or 190 kd (p190) are produced that have deregulated ABL
tyrosine kinase activity. p210 and p190 BCR-ABL oncogenes
contain identical ABL-derived sequences, but they differ in
numbers of BCR-encoded amino acid residues16-19,21,25-28 (Figure 1A). We have previously shown that the BCR-ABL
oncogenes inhibit apoptosis by a Bcl-2 pathway as a part of
their oncogenic phenotype.29 Inhibition of BCR-ABL
expression in Ba/F3+p190 cells reverses this phenotype,
and cells die by apoptosis.3,29
For the design of an M1-GS RNA that can disrupt a chimeric RNA created
by cancer-associated chromosomal translocations, it is necessary to
target the junction sequence. Otherwise, normal mRNA that shares part
of the chimeric RNA sequence will also be cleaved by the M1-GS RNA,
with resultant damage to host cells. In the case of the
BCR-ABLp190 and p210 sequences,
they contain identical ABL-derived sequences, but they differ
in the number of BCR-encoded sequences
(BCR-ABLp190 consists of BCR exon
1 and ABL exon 2, and BCR-ABLp210
consists of BCR exon 3 and ABL exon 2) (Figure 1A). We
have constructed M1-RNA with guide sequences that recognize the
oncogenic messengers at the fusion point (9 nucleotides at each side of
the breakpoint), which we call M1-p190-GS and M1-p210-GS. To test the
effectiveness and specificity of M1-p190-GS and M1-p210-GS, we studied
the in vitro and in vivo effects of these RNA enzymes against both
BCR-ABLp190 and
BCR-ABLp210, bearing in mind the already
mentioned fact that both fusion genes share the same ABL
sequence but differ in that part coming from the
BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as
sequence-specific endonucleases and can exclusively cleave target RNA
that forms base pairs with the guide sequence (GS). The current study
demonstrated that the artificially created ribozyme is specific not
only in vitro but also in cultured cells. This study represents,
to our knowledge, the first gene therapy approach that makes use of the
catalytic activity of M1-RNA to destroy mRNA derived from
the tumor-specific oncogenes created as a result of chromosomal
translocations. The data presented here define a new therapeutic
tool for the treatment of cancer.
Plasmids
Enzymes and chemicals
Ribozyme synthesis The DNA templates for M1RNA with the anti-BCR-ABLp190 or the anti- BCR-ABLp210-directed guide sequences were constructed by the polymerase chain reaction (PCR) with the gene for M1RNA as found in plasmid pTK117 as a template.13 The 5' primer, OliT7 5'-gcgattcTAATACGACTCACTATAG-3', was common to all the ribozymes constructed, annealing with the T7 promoter and providing a 5' EcoRI site for cloning. The 3' primers contained the appropriate guide sequences and were Olip190 5'-gcgtcgacGTGGTGAGACGCAGAAGCCCTTCTATGACCATG-3') and Olip210 5'-gcgtcgacGTGGTAGAGTTCAAAAGCCCTTCTATGAC.CATG-3' The 3' proximal sequences of 10 nucleotides serve as primers for the PCR with the pUC19 sequence. The underlined sequences, the bold sequences, and the lowercase ones correspond, respectively, to the ACCAC-3' sequence, the guide sequences, and the SalI restriction site. The 2 different PCR products (M1-190-GS and M1-210-GS) were cloned in pUC19 and were completely sequenced to exclude any mutation during the PCR reaction. Plasmids pUC19-M1-190-GS and pUC19-M1-210-GS were linearized with SalI and transcribed in vitro with T7 RNA polymerase according to manufacturer's instructions. Transcribed ribozymes were phenol-chloroform extracted and precipitated with ethanol, and their integrity was checked either by acrylamide/urea or agarose electrophoresis.Construction of artificial substrates The fusion points of BCR-ABLp190 and BCR-ABLp210 mRNA were created by annealing the oligonucleotides Oli-190-sense 5'-AGCTTGAGGGCGCCTTCCATGGAGACGCAGAAGCCCTTCAGCGGCCAGTAGCATCG-3') with Oli-190-antisense 5'-AATTCGATGCTACTGGCCGCTGAAGGGCTTCTGCGTCTCCATGGAAGGCGCCCTCA-3') and Oli-210-sense 5'-AGCTTGCCACTGGATTTAAGCAGAGTTCAAAAGCCTTCAGCGGCCAGTAGCATCG-3') with Oli-210-antisense 5'-AATTCGATGCTACTGGCCGCTGAAGGGCTTTTGAACTCTGCTTAAATCCAGTGGCA-3'), respectively. These phosphorylated annealed products, containing 50 nucleotides spanning at the fusion point of BCR-ABLp190 and BCR-ABLp210 (25 nucleotides at each side of the corresponding fusion point) were cloned to the HindIII/EcoRI site of pcDNA3. Once cloned, plasmids were linearized with EcoRI, and artificial substrates were transcribed from the pcDNA3 T7 promoter in the presence of [32P]-GTP and then subjected to purification on 10% polyacrylamide/8 mol/L urea gels. Acrylamide slices containing the transcripts were eluted, ethanol-precipitated, resuspended in water, and were ready for use as targets in the in vitro ribozyme catalytic reaction.Assays for cleavage by M1-GS RNA RNA enzymes and uniformly labeled substrates synthesized as described above were incubated for 4 hours at 50°C in 25 mmol/L Tris, pH 7.5, 50 mmol/L NH4Cl, and 50 mmol/L MgCl buffer in a final reaction volume of 20 µL. Reactions were stopped by the addition of 6 µL of a solution containing 95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF boiled for 5 minutes and chilled on ice. The reaction products were analyzed on 10% polyacrylamide/8 mol/L urea gels.Construction of plasmids for in vivo studies To clone M1RNA-GS to the MFG retroviral vector, plasmids pUC19-M1-190-GS and pUC19-M1-210-GS were digested with EcoRI/SalI and blunt-ended with DNA polymerase I Klenow fragment. The fragment corresponding to the M1-RNA-GS was gel purified and ligated to the NcoI site (blunted) of the pMFG vector. The authenticity of the MFGM1-190-GS and MFG-M1-210-GS constructs was confirmed by DNA sequencing.Cell culture Cell lines used include Ba/F3 cells expressing the human BCR-ABLp190 and 210 proteins, respectively.29,30-32 Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. When required, 10% WEHI-3B-conditioned medium was added as a source of IL-3.Cell transfection Ba/F3 + p190 and Ba/F3 + p210 cells were transfected by electroporation (960 µF, 220 V) with 20 µg MFG-M1-190-GS and MFG-M1-210-GS, respectively, along with 1 µg MC1-puro expression vector. Cell lines were analyzed by Northern blotting for MFG-M1-190-GS and MFG-M1-210-GS expression. Cells were screened for resistance to IL-3 withdrawal and level of BCR-ABL and Bcl-2 expression by Northern blotting. Cell viability was determined by trypan blue exclusion.RNA analysis Total cytoplasmic RNA (10 µg) was glyoxylated and fractionated in 1.4% agarose gels in 10 mmol/L Na2HPO4 buffer (pH 7). After electrophoresis, the gel was blotted onto Hybond-N (Amersham), ultraviolet light-cross-linked, and hybridized to 32P-labeled probes. Loading was monitored by reprobing the filters with mouse -actin cDNA.
Cleavage of model BCR-ABL substrates by M1-GS RNA in vitro The BCR-ABL genes have been well characterized.15-28 These gene products are essential for the viability of tumor cells.3,29,32 Because they are so well studied, we used them as model targets for gene inactivation by M1-GS RNA. DNA encoding GS that contained a sequence of 18 nucleotides complementary to the fusion regions of BCR-ABLp190 and BCR-ABLp210 mRNA was covalently linked to the 3' end of DNA that encoded M1 RNA (Figure 1). To prove the specificity of the constructs M1-p190-GS and M1-p210-GS, we prepared short 50-nucleotide BCR-ABL p190 and p210 substrates that corresponded to the target site indicated by capital letters in Figure 1B. The specificity was tested by incubating the in vitro transcribed M1-p190-GS and M1-p210-GS with the substrates. The RNA transcripts of these constructs, M1-p190-GS and M1-p210-GS, cleaved target RNA that contained 50 nucleotides of the related fusion gene sequence (Figures 2 and 3). Cleavage in the target occurred at the predetermined position. Our M1-GS RNA designed against the product of the BCR-ABL genes only cleaved substrates complementary to the GS. As shown in Figures 2 and 3, M1-p190-GS could only cleave substrate BCR-ABLp190 but not substrate BCR-ABLp210, which contained a sequence of 9 nucleotides identical to the p210 fusion point (Figure 1B). Conversely, M1-p210-GS, which contained a sequence complementary to BCR-ABLp210 mRNA, could efficiently cleave p210 but not p190. Therefore, M1-GS RNA appeared to act as a sequence-specific endonuclease, recognizing substrates through specific base-pairing between the GS and the target sequence in vitro. Moreover, an M1-RNA linked to an unrelated BCR-ABL guide sequence (M1-TC-GS) was unable to cleave p190 or p210 substrates (Figures 2 and 3).
Generation of Ba/F3+p190 and Ba/F3+p210 cell lines transduced with M1-p190-GS and M1-p210-GS RNA Because M1-GS RNA against BCR-ABLp190 and p210 junction sequences acted efficiently and specifically against the related substrates in vitro, we decided to examine the activity of the M1-GS RNA against an endogenous BCR-ABL target. We had previously established murine cell lines, Ba/F3 + p190 and Ba/F3 + p210, that express human p190 and p210 mRNA constitutively by integrating a plasmid construct that express p190 and p210, respectively.3,29,32 Although the parental mouse Ba/F3 cell line is an IL-3-dependent hematopoietic cell line, the transformed Ba/F3 + p190 and Ba/F3 + p210 cells are IL-3-independent because of the activity of p190 and p210 oncogenes.29-31 However, if the expression of p190 and p210 is inhibited, Ba/F3 + p190 and Ba/F3 + p210 cells should become IL-3 dependent and, in the absence of IL-3, they should die.3,29,32 Therefore, during the selection of M1-GS RNA-transduced Ba/F3 + p190 and Ba/F3 + p210 cells, we used 10% WEHI-conditioned DMEM medium as a source of IL-3.Efficient expression of the M1-GS RNAs in mammalian cells To confirm the stable expression of the M1-GS RNA in p190 and p210 cells, we performed Northern blot analysis with a probe that was complementary to the 3'-untranslated region of the MFG vector (Figures 4 and 5). Total RNA from p190 and p210 cells that had been transfected with the various plasmids was extracted after transfection. The results in Figures 4D and 5D clearly demonstrate that each M1-GS RNA was expressed at significant levels and that the transcripts were obviously stable. The finding that M1-GS RNA transcripts in stable transfectants were stable serves to underlie the potential usefulness of our expression system for future gene therapy.
Activity and specificity of the M1-GS RNA against the related endogenous BCR-ABL cellular target We transfected mouse Ba/F3 cells that stably expressed human BCR-ABLp190 and p210 mRNA with plasmid that encoded the M1-GS RNA, and we selected cells by exposure to puromycin 24 hours after transfection. Puromycin-resistant cells were cultured in medium without IL-3, and cell viability was assessed in terms of the ability to exclude trypan blue dye. As shown in Table 1, Ba/F3 + p190 cells that expressed M1-p190-GS died, whereas the control Ba/F3 + p190 cells remained alive 24 hours after the withdrawal of IL-3. Moreover, M1-p190-GS did not kill any Ba/F3 cells that expressed the unrelated BCR-ABLp210 in spite of the fact that both oncogenes contained identical ABL-derived sequences but differed in the number of BCR-nucleotides. This result demonstrated the high specificity of the M1-p190-GS for targeting the chimeric p190 gene. As indicated in Table 1, similar results were obtained with the M1-p210-GS RNA that specifically interfered with the viability of Ba/F3 + p210 cells without affecting the survival of cells expressing the BCR-ABLp190 oncogene.
Gene therapy promises to be an effective strategy for the treatment
of cancer. The molecular characterization of tumor-specific chromosomal
abnormalities has revealed that fusion proteins are the consequence in
most forms of cancer. These fusion proteins are encoded by chimeric
genes generated by chromosomal rearrangements. The presence of the
chimeric molecules is necessary for the persistence of the tumor
cell.3,29,32 These chimeric molecules represent ideal
therapeutic targets because they are unique to the disease state and
they exist in the tumor cells but not in the normal cells of the
patient. Inhibition of chimeric gene expression by antitumor agents
specifically kills the cancer cells without affecting the normal
cells.3,32 As therapeutic agents, zinc-finger
proteins,3 antisense RNA,1,2,7 or
hammerhead-based ribozymes8,9 have been used. However, all
these methods present limitations, implying that new therapeutic tools
are required as gene-inactivating agents that should be able to inhibit
any chimeric fusion gene product.
We thank S. Altman for providing the plasmid pTK117.
Submitted May 19, 1999; accepted September 11, 1999.
Supported by the European Commission (BMH4-CT96-0375); the DGCYT
(UE96-0041, PB96-0816, and 1FD97-0360); the Fundación
Científica of the AECC; the Junta de Castilla y León
(CS12/99); the FIS (99/0935); and the National Institutes of Health (1 R01 CA79955-01). C.C. is a fellow of the Fundación Ferrer
Investigación.
Reprints: I. Sánchez-García, Departamento de
Proliferación y Diferenciación Celular, Instituto de
Microbiología Bioquímica, CSIC/Universidad de
Salamanca, Edificio Departamental, Avenida del Campo Charro s/n,
37007-Salamanca, Spain; e-mail: isg{at}gugu.usal.es.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
70°C.
