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Department of Molecular Biology (Z-D.X., L.O., N.-S.L., J.J.R., Y.F.-Y.) Beckman Research Institute of the City of Hope and Department of Urology (L.O., M.H.K.), City of Hope Medical Center, Duarte, California 91010; and Department of Biochemistry, Physiology, and Medicine (S.M.), Loma Linda University, Jerry L. Pettis Veterans Affairs Medical Center, Loma Linda, California 92357
Address all correspondence and requests for reprints to: Yoko Fujita-Yamaguchi, Ph.D., Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010. E-mail: yyamaguchi{at}coh.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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40%) compared with the level in
parental or M-expressing PC-3 cells. Polymerase II and III
promoter-driven R similarly suppressed IGF-II mRNA levels. Suppression
of IGF-II mRNA levels by R was associated with suppression of IGF-II
protein levels. R- (or RR-) expressing PC-3 cells did not grow under
serum-starved conditions and showed prolonged doubling times in the
presence of 10% FCS compared with those of parental or M-expressing
cells. These results substantiated that IGF-II plays a critical role in
prostate cancer cell growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-II is a 7.5-kDa single-chain polypeptide of 67 amino acid residues, which is processed from its precursor (6). Previous studies suggested that an incompletely processed form of 15-kDa IGF-II is expressed more abundantly than the 7.5-kDa form in many cancers (1, 7, 8, 9). The 15-kDa form of human IGF-II was shown to have a mitogenic potency greater than that of 7.5 kDa (10). We showed that of 36 prostate, 17 breast, 10 bladder cancers, and 9 paraganglioma tissues examined, IGF-II was expressed in more than 50% of prostate, breast, and bladder tumors, and in 100% of paraganglioma tumors (9). Greater expression of the 15-kDa IGF-II relative to the 7.5-kDa IGF-II form was clearly demonstrated in all six prostate cancers and in one of the two breast and two of the four bladder cancers examined (9). The results are consistent with the hypothesis that the 15-kDa form of IGF-II expressed in cancerous cells contributes to autocrine cancer cell growth in vivo.
Evidence is accumulating that an enhanced IGF/IGF-I receptor (IGFIR) signaling leads to increased cancer cell proliferation and tumorigenesis as well as antiapoptotic effects (11, 12). For example, it has been demonstrated in experimental systems that overexpression of human IGFIR promotes ligand-induced neoplastic transformation (13) and tumorigenesis (14) in the presence of an active protein tyrosine kinase. An important role of IGFIR in mediating c-Myc-induced apoptosis of fibroblasts in low serum medium was first reported (15). Since then, a series of studies supporting the antiapoptotic role of IGFIR in cancer cells have been published (16, 17, 18). In brief, reduction of the number of IGFIR by introduction of antisense IGFIR cDNA caused extensive apoptosis in vivo in several transplantable human or rodent tumors (16).
In the case of prostate cancer, it was originally reported that IGF-I is responsible for autocrine growth of human prostate cancer cell lines including androgen-dependent LNCaP as well as hormone-independent DU145 and PC-3 cells (19). We and others more recently showed that IGF-II, but not IGF-I, is produced in those established human prostate cancer cell lines and suggested an autocrine regulation of DU145 and PC-3 cell growth by IGF-II (20, 21, 22). Furthermore, recent studies using in situ hybridization and immunohistochemistry indicated that epithelial cells rather than stromal cells in prostate tumors express IGF-II in vivo (9, 23). These data provided the basis for using prostate cancer as a model to test the hypothesis that cancer cell growth may be regulated by IGF-II in an autocrine manner.
Blockage of IGF-II/IGFIR signaling and subsequent effects on cell
growth, transformation, and tumorigenicity have been reported. Examples
of strategies to block IGF-II/IGFIR signaling include 1) inhibition of
IGF-II expression by antisense oligonucleotides or RNAs (24, 25, 26); 2)
inhibition of IGFIR expression by antisense oligonucleotides or
RNAs (19, 27, 28); 3) blockage of IGFIR by IGFIR monoclonal antibody
such as 
IR-3 (29, 30); and 4) blockage of IGF-mediated growth by
IGF-binding proteins (20, 31). In addition, a number of anticancer drug
agents have been shown to work, at least in part, by suppression of
IGFIR action. For instance, suramin administration to breast, lung, and
prostate cancer patients significantly reduced IGF-I and -II serum
levels (32). Similarly, the antiestrogen tamoxifen has a powerful
cytostatic effect in breast cancer cells, which in part is due to its
inhibitory effect on the IGF-I/IGFIR axis (33).
Ribozymes (RZs) are RNA enzymes that specifically cleave their respective target RNAs, thereby inhibiting the expression of specific gene products. Hammerhead-type RZs work in cis (intramolecularly) in nature, but separation of cis-acting RZs into two RNA fragments can convert them into trans-acting RNAs capable of site-specific cleavage of substrate RNAs. In the last 15 yr, RZs have progressed from an intriguing subject of scientific study to therapeutic agents for the potential treatment of both acquired and inherited diseases (34). It is becoming increasingly evident that RZs can serve the duel function of a tool to elucidate the functional roles of many gene products as well as therapeutic agents designed to functionally destroy deleterious RNAs. Suppression of IGF-II expression by IGF-II-specific RZs in cells has never been reported. We thus constructed catalytically active IGF-II RZs and expressed them intracellularly in human prostate cancer PC-3 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As controls, activity of
RZ was inactivated by introducing a point mutation of G to A in the
catalytic core as shown in Fig. 1. Template DNAs for RZs and mutant RZs
were prepared by filling in the opposite strands of two overlapping
oligonucleotides by PCR. The oligonucleotides used for construction of
the single ribozyme were
5'-ACGCGTCGACCAGCATCCT(A/G)ATGAGTCCGTGAGG-3' (34
bases) and 5'-GCTCTAGAGCGGGGAAGTTTCGTCCTCACGGACTC-3' (35
bases), which contained restriction enzyme SalI and
XbaI sites, respectively. The primers for the double RZs
were
5'-ACGCGTCGACAGAAGGTCT(A/G)ATGAGTCCGTGAGGACGAAAGAAGCACCAGCAT-3'
(53 bases) and 5'GCTCTAGAGCGGGAAGTTT
CGTCCTCACGGACTCAT(C/T)AGGATGCTGGTGCTT-3' (52 bases), which
contained restriction enzyme SalI and XbaI sites,
respectively. For both single and double RZs, the sequences that are
complementary are underlined. Note that the position
indicated as (/) represents equal ratios of both nucleotides
incorporated to generate both active and inactive (mutant) RZ at the
same time. The template DNAs for single and double RZ were prepared by
five cycles of PCR of 94 C for 1 min, 37 C for 1.5 min, and 72 C for
1.5 min, and five cycles of PCR of 94 C for 1 min, 33 C for 1.5 min,
and 72 C for 0.5 min, respectively. After the PCR, the quality of PCR
products was examined by PAGE. The PCR products were cleaned up,
digested with SalI and XbaI, and subcloned into
the SalI/XbaI sites of pTZU6 + 27 vector, which
contained the human U6 promoter and pUC19 multiple cloning site (36).
After the ligation reaction was carried out, the ligation products were
electroporated into Escherichia coli XL-1 blue cells. The
bacteria were plated on the LB agar containing ampicillin, X-gal, and
isopropyl-ß-D-thiogalactopyranoside. Plasmids were
prepared from white colonies, and colonies containing the correct
inserts were identified by SalI/XbaI digestion
and gel electrophoresis. To confirm authenticity of the RZs, plasmid
DNAs were prepared and subjected to Southern blot analysis and DNA
sequencing. Clones expressing single RZ (R) and its mutant (M, an
inactive RZ in which a point mutation of G5 to A is introduced), and
double RZ (RR), its mutant (MM), and double RZ with one mutant, RM or
MR, were isolated. These plasmids, pTZU6+27/RZ, were used for both
in vitro transcription of RZ as well as RNA polymerase (pol)
III-driven expression of the RZ in mammalian cells.
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Subcloning of IGF-II substrate
To test the in vitro cleavage efficiencies of the
RZs, a substrate human IGF-II RNA [-6 to +74, 80 nucleotides (nt)]
was prepared by subcloning a small fragment of IGF-II cDNA into
pBluescriptII KS(+) (Stratagene, San Diego, CA). A
DNA template for the IGF-II RNA was prepared by PCR from the
full-length IGF-II cDNA (9). The primers used for the PCR were:
5'-CGGAATTCCGACACCAATGGGAATCCC-3' and 5'-CGGGATCCCGGCAGGC
AGCAATGCAGCACGA3-', which contained a restriction enzyme site,
BamHI and EcoRI, respectively. PCR was performed
as follows: 95 C for 3 min, and then 30-cycle amplification of 95 C for
1 min (denaturing), 55 C for 1 min (annealing), and 72 C for 1 min
(extension).
The PCR product, digested with BamHI and EcoRI, was ligated into pBluescript KS. Transformation of the ligation mixture was achieved by electroporation. Briefly, 3 µl of the ligation mixture were electroporated into E. coli XL1 blue cells. After LB was added, the bacteria were incubated at 37 C for 1 h to allow the antibiotic-gene express, and then plated on LB/1.5% agar containing 100 µg/ml Ampicillin, which had been layered with 100 µl of 0.05% X-gal and 75 mM IPTG, and grown at 37 C overnight. Plasmids were prepared from white colonies and their inserts were examined by BamHI/EcoRI double digestion and gel electrophoresis. Colonies containing the right-size insert were selected. To confirm the authenticity of the IGF-II substrate sequence, plasmid DNAs were subjected to DNA sequencing.
Preparation of IGF-II mRNA substrate by in vitro transcription
The plasmids that contained the IGF-II substrate (-6 to +74)
and the full-length IGF-II were linearized by BamHI
digestion and used as templates for in vitro transcription.
Transcription reactions were carried out at 37 C for 1 h in 40
mM Tris-HCI buffer, pH 7.9, containing 0.2 µg DNA
template, 0.5 U/µl of T3 RNA pol, 20 mM
MgCl2, 10 mM NaCl, 10 mM
dithiothreitol, 0.5 mM each of ATP, GTP, and uridine
triphosphate, 0.05 mM cytidine triphosphate (CTP), 10 µCi
of [-32P]CTP, and 1 U/µl of RNasin (Promega Corp., Madison, WI). After transcription, the RNAs were treated
with RNase-free DNase I for 15 min and purified by electrophoresis in a
6% denaturing polyacrylamide gel for 1 h at 200 V. Before
purification, a small aliquot of RNAs was removed to use for
calculation of the specific activity. After electrophoresis, the gel
was exposed to a Kodak XRP film (Eastman Kodak Co.,
Rochester, NY) for 1 min and the film was developed. The region of the
gel containing the desired RNA band was excised and crushed into fine
pieces. The IGF-II substrate RNA was eluted in an elution buffer
overnight. The aqueous phase was removed and mixed with
phenol-chloroform-isoamyl alcohol (25:24:1) to extract the RNA. The
substrate RNA was precipitated with ethanol, redissolved in 20 µl
diethyl pyrocarbonate-treated H2O, and stored at
-75 C.
In vitro transcription of RZs
The pTZU6+27/RZ plasmids were linerarized by XbaI
digestion and used as templates for transcription of IGF-II RZ as
described for substrate RNA preparation with two exceptions: only a
trace amount of the radioisotope was used and T7 RNA pol was used
instead of T3 RNA pol.
In vitro RZ cleavage assays
RZ assays were performed according to the methods previously
described (37, 38). Briefly, RZs and substrate were heated
independently for 1 min at 90 C in 10 µl of water. After cooling to
25 C, the reaction buffer was added to a final concentration of 10
mM MgCl2, 140 mM KCl, and 50
mM Tris-HCl, pH 7.5. RZs and substrate were then combined
and incubated at 37 C. The reaction was stopped by adding an equal
volume of stop solution (0.5% of SDS/25 mM EDTA) and then
100 µl of phenol. The aqueous phase was brought to 100 µl. After
vortexing, the aqueous phase was removed and the RNAs were precipitated
with ethanol. The RNAs were analyzed by 6% polyacrylamide/8
M urea gel electrophoresis. Radioactive bands were
visualized by autoradiography and quantitated by a PhosphorImager
(Molecular Dynamics, Inc.).
The single turnover experiments using RZ excess over substrate were carried out to determine the first-order rate constant for cleavage of the substrate (39). The initial cleavage velocities under single-turnover conditions were determined at a constant substrate concentration of 1 nM and varying RZ concentrations. Reaction mixtures (10 µl) containing 0, 2.5, 5, 10, 20, 30, and 40 nM RZ and the substrate RNA (at a final concentration of 1 nM) were incubated at 37 C for 2 h. The reaction was stopped by the addition of 10 µl of the stop solution and analyzed by PAGE as described above. Kcat/Km values were obtained by plotting the remaining fraction of the 32P-labeled substrate RNA (Frac S) against the ribozyme concentration ([RE]) according to the following equation k = - ln (FracS)/t = [RE] x kcat/Km where k is the observed reaction rate and t is the reaction time of 2 h.
Cell culture and transfections
Human prostate cancer PC-3 cells were transfected with
pTZU6+27/RZ or pcDNA3/RZ. The calcium-phosphate precipitation method
was used to cotransfect PC-3 cells with pTZU6+27/RZ expression and neo
vectors (40). Briefly, 24 h before transfection, exponentially
growing cells were harvested by trypsinization and replated into 90-mm
tissue culture dishes. Ten milliliters of RPMI 1640 medium with 10%
FCS were added, and cells were incubated overnight at 37 C in a
humidified incubator in an atmosphere to 5% CO2. To those
cells, were added 0.5 ml of 0.25 M CaCl2
containing 18 µg of superhelical plasmid RZ DNA, 2 µg neo Vector
DNA, and 0.5 ml of 2x PBS-buffered saline. The cells were incubated
for 15 min at room temperature. RPMI 1640 medium with 10% FCS was
added dropwise to the cells in the dishes, which were gently swirled.
The cells were incubated for 16 h at 37 C in a humidified
incubator in an atmosphere of 3% CO2. The medium was
removed by aspiration, and cells were rinsed twice with medium. Ten
milliliters of fresh medium were added, and cells were incubated for
24 h at 37 C in a humidified incubator in an atmosphere of 5%
CO2. After 24 h incubation in nonselective medium to
allow expression of the transferred genes to occur, the cells were
trypsinized and replated in medium containing 600 µg/ml of G418. The
medium was changed every 3 days for 4 weeks to remove the debris of
dead cells and to allow colonies of G418-resistant cells to grow.
For the RNA pol II-driven RZ expression system, PC-3 cells were transfected by electroporation. Cells were grown to 70% confluence, trypsinized for 2 min, centrifuged in the table-top centrifuge, and resuspended in PBS buffer at a cell density of 5 x 106 cells per ml. This suspension was preincubated with 5–20 µg of DNA in 800 µl of PBS buffer on ice for 10 min with mixing, transferred to a cuvette, and immediately pulsed with the following settings: capacitance, 800 µFarads; resistance (R4), R4 (72 ohm); and charging voltage, 350 V. A 4-mm gap chamber was used in a BTX electroporator (San Diego, CA). After pulsing, the cells were left in ice for 10 min, transferred into the Petri dish, and cultured in the medium containing 600 µg/ml of G418 for at least 1 month.
Detection of IGF-II RZs expressed in PC-3 cells
RZ expression in G418-resistant clones was confirmed by
RT-PCR. A set of primers for detection of RZ prepared were 5'-primer,
5'-TCGCTTCGGCAGCACGTCGAC-3', containing a sequence corresponding to the
junction between U6 promoter and the RZ, and 3'-primer,
5'-GGGAAGTTTCGTCCTCACGGA-3', containing a sequence of the
catalytic stem underlined. RT reaction was performed
at 37 C for 45 min by mixing 4 µg of total RNA from the PC-3
transformants with 10 pmol of the 3'-primer in 50 mM
Tris-HCl, pH 7.5, containing 10 mM dithiothreitol, 75
mM KCl, 3 mM MgCl2, 1
mM of each deoxynucleoside triphospate, 800 U of Moloney
murine leukemia virus reverse transcriptase, and 20 U of RNasin. RT
reaction products, equivalent to 1 µg of total RNA from each clone,
were processed through 30 cycles of PCR with denaturation at 94 C for 1
min, annealing at 47 C for 2 min, and synthesis at 72 C for 3 min, in a
final volume of 20 µl. One half of the reaction product was analyzed
in 2% agarose gel, stained with EtBr, and blotted onto a nylon
membrane. The blot was hybridized with a specific
32P-labeled oligonucleotide probe, 5'-TGGTC
GTAGGACTACTCA-3' (underline and bold
indicate the complementary sequence to the catalytic stem and the
position mutated in M, respectively) at 54 C for 16 h in 6x SSC,
5x Denhardt’s solution, 1% SDS, and 100 µg/ml herring-sperm DNA.
The membrane was washed three times in 2x SSC and 0.1% SDS for 30 min
at 51 C. The bands hybridized with the ribozyme-specific probe were
visualized by autoradiography. For detection of RZ in pcDNA3/RZ clones,
PT-PCR was performed under the same conditions as above using
5'-primer, 5'-CCCACTGCTTACTGGCTTATCGA-3', and 3'-primer,
5'-GGACAGTGGGAGTGGCACCTTC-3'.
Quantitation of IGF-II mRNA levels in PC-3 cells by quantitative
competitive (QC)-PCR
The 5'- and 3'-primers used to detect RZ-mediated cleavage of
IGF-II RNA in cells were 5'-CCAGCACCAATGGGAAT CCCAATGGGGAAG-3' (-10 to
+21) and 5'-GTATCTGGGGAAGTTGTCCGGAAGCAC GGTC-3' (+294 to +324),
respectively. pBluescript KS/IGF2, pBluescriptII KS(+)
(Stratagene, La Jolla, CA) containing approximately 1 kb
EcoRI fragment encoding the human precursor IGF-II
(911–2067 nt) (9) was used to generate a new plasmid that encodes a
competitor IGF-II sequence. Detailed methods will be published
elsewhere (Xu, Z.-D., R. Mineo, S.-J. Liang, and Y. Fujita-Yamaguchi,
manuscript in preparation). Briefly, a 110-bp
SalI/SalI fragment was inserted into the IGF-II
sequence between the PCR primers so that when the competitor IGF-II RNA
was transcribed, it would provide a template for a PCR product that is
110 bp longer than that derived from the authentic IGF-II mRNA. In the
presence of the competitor RNA, a 444- bp band, which was therefore
produced in addition to the 334-bp band derived from an endogenous
IGF-II RNA.
Conditioned medium
PC-3 cells and transfectants were seeded in 25-cm2
flasks in the regular medium. The next day, cells were washed with PBS
three times and grown for 2 days in 4 ml of RPMI 1640 containing 0.1%
BSA. Condition media were collected and analyzed for IGF-II and -I as
previously described (22). At the time of conditioned medium
collection, cell numbers were determined with a hemocytometer, and the
IGF-II protein level was expressed as nanograms/ml/105
cells.
Determination of cell proliferation
Cell growth was determined by the
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method, which measures mitochondrial dehydrogenase activity that is
active only in living cells. Cells (104 cells per well)
were plated in 96-well plates containing 100 µl of medium and
cultured 37 C for 4 days. At days 1, 2, 3, and 4, 20 µl of 0.5% MTT
were added to the wells in triplicate. After incubation at 37 C for
4 h, the medium was removed and 100 µl of isopropyl alcohol
supplemented with 0.05 N HCl were added. The color
developed was quantitated by measuring absorbance at 540
nM.
Doubling times of parental PC-3 cells and transfectants were determined by time-lapse microscopy. Approximately 105 cells of each clone were seeded in medium supplemented by 10% FCS in a T-25 flasks and incubated at 37 C overnight. The flasks were placed under a phase contrast microscope (Nikon, Melville, NY) in a warm room. A good view of isolated cells was selected and video-recorded for 4 days. Doubling time for each clone was averaged from those of five to eight cell divisions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Time course. The substrate, 7.7 nM, was incubated
with 12.3 nM RZ, R or M, at 37 C for 10 to 210 min
(substrate-RZ ratio of 0.6:1). As shown in Fig. 2, only the active RZ cleaved the
substrate in a time-dependent manner. After incubation for 210 min, all
the substrate was apparently digested by R.
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In vitro cleavage reactions of single and double RZs. RZ
activities of single RZ (R), double RZ (RR), its mutant (MM), and
double RZs with one mutant (RM and MR) were analyzed. Figure 3A demonstrates that in vitro
cleavage reactions of both single and double RZs produced the expected
sizes of cleavage products as illustrated in Fig. 3C. Mutant double RZ
(MM) did not cleave the substrate RNA at all. To compare relative
cleavage efficiencies among various RZ constructs, the intensity of the
product, either P1 or P1', was quantitated. Figure 3B clearly indicates
that R and RM are equally active whereas MR is less effective in
cleaving the substrate IGF-II RNA than RM or R. The double RZ, RR,
appeared the most active RZ of the four RZs that we constructed to
target IGF-II RNA.
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), catalytic efficiencies of
single and double RZs were further compared.
Kinetic analysis for the two RZs, R and RR, were performed under
single-turnover conditions using the short IGF-II RNA substrate. The
results summarized in Fig. 4 revealed
Kcat/Km = 1546 and 4772
M-1s-1, respectively. This indicates that the
double RZ is approximately 3-fold more efficient in cleaving the
substrate IGF-II RNA than the single RZ in vitro
(P < 0.05).
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, lanes 11 and 12). The time course
experiment, however, revealed that during the first 3 h of
incubation, RR cleaved the substrate in a time-dependent manner whereas
R did not appear significantly to cleave the substrate (Figs. 5A, lanes
1–8, and Fig. 5B). This result is consistent with that of the kinetic
analysis that indicated that RR is more active than R in
vitro.
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Stable expression of RZs in PC-3 cells driven by RNA Pol III
promoter (pTZU6+27 vector).
Detection of RZ mRNA in PC-3 cell transfectants.Expression of R, M, and RR in four, two, and two transfectants,
respectively, was confirmed by EtBr-stained agarose gel electrophoresis
of RT-PCR products prepared from DNase I-treated RNA (data not shown).
No PCR amplification was made when PCR was performed from the
corresponding RNA preparations without RT (data not shown). RT-PCR
products derived from single and double RZs migrated at the same
positions as PCR controls for R and RR, respectively, prepared from the
plasmids encoding R and RR (data not shown). The authenticity of R, M,
and RR was confirmed by Southern blot analysis using an oligonucleotide
probe complementary to the R sequence (data not shown). The mutant RZ
did not hybridize with the R-specific probe at 54 C due to a one-base
mismatch in the probe sequence.
Quantitation of IGF-II mRNA levels in PC-3 cell
transfectants.IGF-II mRNA levels in parental and transfected PC-3
cells were determined by QC-PCR using a competitive IGF-II RNA as a
control (Fig. 6A). Endogenous IGF-II mRNA
levels in PC-3 cells expressing R or RR were suppressed by 62% or
68%, on average, compared with that of parental PC-3 cells, whereas
cells expressing M had little effect (12% suppressed) on the IGF-II
mRNA levels (Fig. 6, A and B) as determined by quantitative RT-PCR.
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), indicating
that catalytically active RZ expression is inhibitory for PC-3 cell
growth. These results suggest that cleavage of IGF-II mRNA by R or RR,
but not the potential antisense effect of M per se, is
required for inhibition of PC-3 cell growth.
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Detection of RZ mRNA in PC-3 cell transfectants.Of
10 transfectants examined, expression of R was confirmed in 4
independent clones by RT-PCR (Fig. 7A).
EtBr-stained agarose gel electrophoresis of the RT-PCR product also
identified six G418-resistant clones that do not express R. They were
used as vector-control cells below.
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). Endogenous IGF-II mRNA
levels for parental, R-expressing, and vector-transfected PC-3 cells
were 0.2 (n = 1), 0.045 ± 0.005 (n = 4), and 0.122
± 0.018 (n = 6), respectively. Endogenous IGF-II mRNA levels in
R-expressing PC-3 cells were thus reduced to approximately 40% level
when compared with those of six transfectants that do not express R
(vector-control), and to about 20% level when compared with that of
parental PC-3 cells. In parallel to the IGF-II mRNA levels, IGF-II
protein levels secreted into conditioned media by R-expressing PC-3
cells were significantly lower than those of parental or vector clones,
while that of the vector-transfected cells was modestly reduced (Table 2)2.
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, were analyzed in SFM (Fig. 8A) or in the presence of 2% FCS (Fig. 8B). The results clearly demonstrate that PC-3 cells expressing the
IGF-II RZ do not grow well under serum-free or 2% FCS conditions. The
results are similar to the observation we had for R- or RR-expressing
PC-3 cells under the control of RNA pol III promoter. Since the
R-expressing PC-3 cells were isolated after culturing those
transfectants in the medium containing 10% FCS, they must have been
able to grow in the presence of 10% FCS (see time-lapse microscopy
experiments below).
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). This result indicates that PC-3 cells
expressing the IGF-II RZ showed a significantly prolonged doubling
times compared with parental PC-3 cells, while the doubling times of
vector-transfected PC-3 cells are not significantly different from
those of the parental cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RM > MR. This indicates that the first RZ cleavage site may
be a better target than the second RZ cleavage site since the cleavage
of the former by R, RM, or RR was more efficient than that of the later
by MR. Of three double RZs examined, RR was more active than RM and MR.
RR was also 3-fold more efficient in cleaving a substrate IGF-II RNA
than R as judged by kinetic analysis under single-turnover conditions.
Both RM and R were equally active despite the difference in the length
of antisense arms, 29 and 17 nt, respectively. These results suggest
that the tandem repeat of two catalytic domains enhances the catalytic
efficiency of the RZ rather than the longer antisense arms. When a
longer RNA substrate was used, RR was much more effective in cleaving
the substrate than R. However, the cleavage of the longer substrate
(1157 nt) required a much higher concentration of RR than that of the
shorter substrate (147 nt). These observations are generally in good
agreement with the results of previous studies that systematically
analyzed the effects of catalytic core tandem repeats (42) or antisense
arm lengths on RZ cleavage reactions in vitro (43).
Single and double RZs were stably expressed in human prostate cancer
PC-3 cells under the control of RNA pol III or pol II promoter. Stable
transfectants were isolated and used to determine the efficacy of the
RZ activity in cells and effects of active RZs on cell growth.
Establishment of quantitative assays for the target RNA was necessary
for the detection of RZ cleavage reactions in vivo (in cell
line). The QC-PCR method was used in this study because it is very
sensitive, specific, and quantitative (44). In contrast to most of the
RT-PCR methods that use two pairs of primers, the competitor IGF-II RNA
shares the same sequences for primer binding as the target endogenous
IGF-II mRNA. But, it contains an additional 110-base long insert that
will give a PCR product larger than the PCR product generated from the
target endogenous IGF-II mRNA. This 110-base difference in size was
sufficient for easy separation of the two PCR products on denaturing
polyacrylamide gels and for quantitation of the radioactive bands with
a PhosphorImager (Fig. 6A). Using QC-PCR, we have clearly demonstrated
that the intracellularly expressed RZs cleaved endogenous IGF-II
mRNA.
Interestingly, in our study, single and double RZs showed almost the same level of efficacy in reduction of IGF-II mRNA in PC-3 cells. This observation can be explained in the following ways: 1) due to unique secondary or tertiary structure caused by other cellular factors, interaction of IGF-II mRNA with the double RZ may make one of the cleavage domains unable to anneal with the substrate (second cleavage site); thus there is no cleavage reaction for that domain; or 2) the RZ-expressing clones selected may be only those that are able to moderately reduce IGF-II to a point that does not cause cell death.
Although in vitro studies of RZs are essential steps for quantitating their catalytic efficiencies, they cannot be used to predict the efficiency of the RZs when expressed in cells. Due to the complexity of eukaryotic gene expression, and also multiple factors involved, the RZ action in the cell is heavily influenced by the cellular environment. Factors that may interfere with RZ action in the cell include RZ expression level, colocalization of RZs with the target RNAs, location of the RZ gene relative to the target gene in the host genome, target sites at which the RZ functions, secondary structure of the target RNA or RZs, and cofactors or inhibitors such as proteins, Mg2+ concentration, and pH. For example, if RZ expression is not highly efficient, the level of RZs may be too low to cleave the target RNA. Even though RZs are efficiently expressed, if RZs are not in the same cell compartment with the target RNA, RZ activity would be dramatically decreased.
The secondary structure of the target RNA or RZs is also very important. Since RZs cleave the target RNA by first complementary binding to the target RNA, if the secondary structure of target RNA or RZs interferes with the binding of RZ and target, RZ activity will be reduced. Also, proteins may bind to portions of the RNA substrate or to the RZ itself and may facilitate or interfere with the catalysis. For example, previous studies demonstrated an enhancement of RZ cleavage reactions by RNA-binding proteins such as heterogeneous nuclear ribonucleoprotein A1 and the capsid protein NC7 of human immunodeficiency virus type 1 (HIV-1) (37, 45, 46). In our study, pTZU6 + 27 and pcDNA 3 vectors were used to express IGF-II RZs intracellularly under the control of RNA pol III and II, respectively. The RZ transcripts from pTZU6+27/RZ should be relatively stable and expressed in both nucleus and cytoplasm, but mainly in the nucleus (36, 47, 48). Pol III promoters, which naturally drive transfer RNA and small nuclear RNA synthesis, are good for high-level, non-tissue-specific expression of short RNAs. For example, Thompson et al. (41) used a transfer RNA-based RNA pol III promoter-driven RZ and found high accumulation of recombinant pol III RZ transcripts in all cell lines tested. The RZ activity was readily detectable in total RNA extracted from stably transformed human T cell lines. Michienzi et al. (48) used U1 small nuclear RNA chimeric RZs with substrate specificity for the Rev pre-mRNA of HIV and showed that this construct caused more efficient reduction in the Rev pre-mRNA level in vivo. Kawasaki et al. (49) reported that by using a pol III-driven vector, the adenoviral-E1A-associated 300-kDa protein expression in HeLa cells was inhibited. In contrast, the RZ transcripts from pcDNA3/RZ can be ubiquitously expressed in the cytoplasm under the control of RNA pol II. Pol II promoters, including viral promoters, which are used naturally for mRNA synthesis, can allow tissue-specific expression of RZs. A disadvantage of Pol II promoters is their requirement for long coding sequences, so that the RZ sequence may be placed in long RNA molecules. This can interfere with RZ conformation.
In the present study, both pol II and pol III promoter-driven vectors
similarly expressed RZs and suppressed IGF-II mRNA levels in PC-3
cells, which resulted in cell growth inhibition. It should be noted
that the growth of M-expressing PC-3 cells was not affected although
the M RZ could have an antisense effect. Since the in vitro
experiments revealed formation of a stable IGF-II RNA substrate/M RZ as
seen in Fig. 2, formation of the complex in cells might also take
place. Our results, however, indicate that if such a complex formed in
PC-3 cells, it was not sufficiently stable to inhibit cell growth. It
is possible that short RNA hybrids may be destroyed by ribosome
translocation or heterogeneous nuclear ribonucleoproteins (37).
The different results in the growth effects observed between R and M
RZs thus support the notion that the catalytic activity of R RZ is
required to significantly inhibit cell growth under our experimental
conditions.
Suppression of IGF-II mRNA levels by the RZs was associated with reduced IGF-II protein levels. The reduction of protein levels was, however, not as significant as that of mRNA levels. This suggests that there may be a possible difference in the regulation of two IGF-II forms, the regular 7.5-kDa form and the incompletely processed 15-kDa form. The IGF-II protein levels measured in this study reflect those of both 7.5- and 15-kDa IGF-II since the RIA used an anti-IGF-II antibody that binds both forms of IGF-II. It is possible that the expression of the 15-kDa IGF-II, which is commonly expressed in cancerous tissues, is more affected by IGF-II RZ actions while the 7.5-kDa IGF-II level may not be affected. Further analysis of the expressed IGF-II protein by immunoblotting is necessary to substantiate this possibility. It is also necessary to examine whether expression of other IGF system components including IGF-binding proteins has been changed when an active RZ is intracellularly expressed.
RZ-expressing PC-3 cells did not grow well but rather die under
serum-starved conditions while they grew in the medium supplemented by
10% FCS, which was the condition used for isolation of the
RZ-expressing clones. It has been shown that PC-3 cells undergo
apoptosis under serum-free conditions (50), and that IGFs are
antiapoptotic (15, 16, 17). Consistent with those previous studies, the
results shown in Fig. 8 show that PC-3 cell and vector-control cells
seem to undergo apoptosis in SFM, and that the reduction of IGF-II
levels by intracellular expression of IGF-II RZs seem to stimulate
apoptosis of PC-3 cells in SFM and low serum medium. Investigation on
mitogenic and apoptotic pathways of R-expressing PC-3 cells are now in
progress.
In conclusion, the present study demonstrated that IGF-II RZs were active at least in one cell line and were able to lower endogenous IGF-II mRNA and protein levels, and that blockage of IGF-II/IGFIR signaling by IGF-II RZs inhibited cell growth. The IGF-II RZ can thus be used as a tool to investigate the role of IGF-II in other physiological systems. The study also provided the basis for the development of IGF-II RZs as future cancer therapeutics.
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2 IGF-I protein levels secreted into conditioned
media by parental and PC-3 cell transfectants were determined. The
IGF-I levels measured, however, were too low to be informative given
that PC-3 cell IGF-I levels are below accurate levels of detection
(22 ).
Received August 27, 1998.
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. To produce an
inactivated RZ, a highly conserved base, G5, in the catalytic domain,
which is required for cleavage, was mutated to A as shown (G 
A).
),
vector-control PC-3 cells (V37; 
), R-expressing PC-3 cells (R4; 
,
R6; 
, and R39; 
) were cultured either in serum-free medium (SFM)
or in the presence of 2% FCS. Cell growth was determined for 4 days in
triplicate by the MTT method. Growth curves are normalized by setting
the OD value of day 1 as 100% for each transfectant. Shown are the
representative results from three independent experiments.