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Department of Cancer Biology, Lerner Research Institute [Y. X., Z. W., J. M., S. P., G. C., R. H. S.], and Urological Institute [E. A. K.], Cleveland Clinic Foundation, Cleveland, Ohio 44195; Translational Genomics Research Institute, Phoenix, Arizona 85004 [J. D. C., J. M. T.]; and Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287 [W. B. I.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2; Ref. 3
]. The only well-established function of 2–5A is the activation of RNase L. Although 2–5A compounds generally vary in length from two to five or more adenylyl residues, the principal products observed in IFN-treated and virus-infected cells are the inactive diadenylate, ppp5'A2'p5'A, and the active tri- and tetra-adenylate species [ppp5'A(2'p5'A)2 and ppp5'A(2'p5'A)3; Ref. 4
]. These oligonucleotides are unusual in nature because even though nonenzymatic synthesis of nucleic acids favors 2',5' linkages, the vast majority of internucleotide linkages formed enzymatically are 3',5', including mature species of RNA and DNA (5)
. Exceptions are 2' to 5' junctions in lariat RNA splicing intermediates, a 2',5'-phosphodiester linkage in DNA-RNA complexes known as multicopy single-stranded DNAs, and 2–5A (3
, 6 , 7)
. Because 2–5A is the only known nucleic acid with two or more consecutive 2',5' linkages it occupies a unique place in nucleic acid biology by providing an unambiguous signal to RNase L, resulting in cleavage of single-stranded RNA. A large number of 2–5A analogues has been described previously with modifications of the bases, riboses, internucleotide linkages, 5'-phosphoryl groups, and 2',3' termini (reviewed in Ref. 5
). Nevertheless, development of biostable and potent small molecule activators of RNase L that are convenient to synthesize and purify have remained elusive. Natural 2–5A molecules are very efficiently degraded by a combination of 5'-phosphatase and 2',5'-phosphodiesterase activities present in cells and in serum. Therefore, stabilization of the 2–5A molecule to catabolic enzymes while retaining RNase L activation ability was an important goal in the present study.
RNase L, the target of 2–5A, is a 741-amino acid protein (human form) with a bipartite domain structure in which the N-terminal half represses the RNase domain in the COOH-terminal region (Fig. 1
; Ref. 8
). The repressor half consists of nine ankyrin repeats. 2–5A binding to the repressor region of RNase L relieves the inhibition caused by the ankyrin repeats, presumably as a result of inducing a conformational change in the enzyme that unmasks the dimerization and RNase domains (9)
. The seventh and eighth ankyrin repeat contain a duplicated P-loop-like motif (GKT) implicated in mediating binding to 2–5A (10)
. RNase L also contains several protein kinase-like domains in its COOH-terminal half. The kinase-like and RNase domains of RNase L are homologous to the Ire1 proteins, with both kinase and endoribonuclease activities, that function in the unfolded protein response (9)
.
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A, resulting in a methionine to isoleucine missense mutation in the translational start codon, thus preventing translation (1)
; 795GT, converting a glutamic acid within the 2–5A binding domain of RNase L to a stop codon (E265X; Refs. 1
and 11
); and 471
AAAG, causing a frameshift at codon 157 and a translation stop after seven additional codons (Refs. 12
and 13
; Fig. 1
). Loss of the wild-type RNASEL allele in prostate tumor tissue was reported in cases with either the 795GT (E265X) or 471AAAG mutation (1
, 12)
. In addition, there exists among prostate cancer cases and controls several missense variants in RNase L (Fig. 1
; Refs. 1
, 11
, and 14, 15, 16
). Among these, only R462Q has been thus far associated with increased prostate cancer risk (11
, 15)
. For example, we recently implicated the R462Q variant of RNASEL (1385GA) in unselected (including both familial and nonfamilial) prostate cancer cases (15)
. An expanded study was performed on DNA isolated from 423 unselected prostate cancer cases and 454 unaffected sibling controls. A significant association of the R462Q variant with cases was observed (P = 0.011). The odds ratios indicated that carrying one copy of the R462Q variant gene increased the risk of prostate cancer by 
1.5-fold, whereas having two variant alleles doubled the risk. Results implicated R462Q in up to 13% of cases, which if confirmed would make it the most prevalent genetic marker for prostate cancer. Therefore, R462Q could be an important risk marker for prostate cancer in the general male population. Here, we have investigated the impact of RNase L activated by novel 2–5A analogues on apoptosis of the human prostate cancer cell lines PC3, DU145, and LNCaP, derived from metastases of brain, bone, and lymph node, respectively. Results show that 2–5A analogues have the ability to induce apoptosis of such late-stage prostate cancer cells. In addition, among eight naturally occurring missense variants evaluated, only R462Q significantly decreased RNase L activity. The deficiency in the R462Q variant was correlated to a decrease in enzyme dimerization to the active form and a reduction in the ability to cause apoptosis. Our findings support the association of RNase LR462Q with prostate cancer risk while also highlighting the ability of RNase L activators to induce death of prostate cancer cells derived from late-stage tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oligonucleotide Synthesis.
Chemical syntheses of the oligonucleotides were performed on 1-µmol columns (Glen Research) using an ABI model 380B automated DNA synthesizer (Applied Biosystems). After syntheses, the columns were removed from the instrument and the supports were transferred to screw-capped vials (4 ml; Wheaton, Millville, NJ). The supports were treated with 3 ml of ammonium hydroxide/ethanol solution (3:1, v/v) for 2 h at room temperature and then at 55°C for 8 h. The solutions were dried in vacuo on a Savant Speed-Vac. The residues were treated with 1 M tetrabutylammonium fluoride in tetrahydrofuran solution (Aldrich, St. Louis, MO) overnight. Tetrahydrofuran was removed using Speed-Vac. Compounds were dissolved in 1 ml of water and vortex mixed. The crude products were purified by HPLC3
on a polystyrene reverse phase column (PRP-100; Hamiliton Co., Reno, NV). Solvent A was 10 mM tetrabutylammonium dihydrogenphosphate (pH 7.5) in water, and solvent B was 10 mM tetrabutylammonium dihydrogenphosphate (pH 7.5) in acetonitrile:water (8:2, v/v). Elution was with a convex gradient of 5–80% solvent B in solvent A in 60 min at a flow rate of 1.5 ml/min. Fractions containing the compounds were pooled, dried, and then desalted on Sep-Pak columns (Waters Corp., Milford, MA), using 80% methanol as eluent. The tetrabutylammonium salt was transformed into the sodium salt by ion-exchange using Dowex 50W (sodium form; Bio-Rad, Hercules, CA).
Natural 2–5A [p3(A2'p)nA, where n = 1 to 3] was prepared enzymatically from ATP using hexahistidine-tagged and -purified recombinant porcine Mr 42,000 2–5A synthetase (a gift from R. Hartmann, Cleveland Clinic, Cleveland, OH; Ref. 17
). Individual 2–5A oligomers were purified using reverse-phase HPLC.
Determining the Stability of the 2–5A Analogues in Human Serum.
The stability of 2–5A compounds (each at 47.8 µM) was determined by incubation in 700 µl of human serum (Sigma-Aldrich, St. Louis, MO), diluted to 800 µl with water at 37°C. Aliquots (100 µl) were removed at 0, 1, 2, 4, 7, and 24 h, heated to 100°C for 5 min, centrifuged 10 min at 10,000 x g at 2–4°C, and the supernatant removed for HPLC analysis. 2–5A compounds and their degradation products were analyzed by HPLC on an Ultrasphere ODS column (4.6 x 250 mm) (Beckman).
Cell Culture and Transfections.
Hey1B (human ovarian carcinoma), DU145, PC3, and LNCaP cells were grown in RPMI 1640 supplemented with streptomycin-penicillin and 10% heat-inactivated fetal bovine serum. L929 (mouse fibroblast) and HeLa M cells (human cervical epithelial cells) were grown in DMEM supplemented with streptomycin-penicillin and 10% heat-inactivated fetal bovine serum. Mouse JM03 cells were isolated from a spontaneous rhabdomyosarcoma from RNase L-/- p53-/- mice.4
Transfection of 2–5A compounds was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, cells were plated 1 day before transfection, so that the cells are 70–80% confluent at the time of transfection. 2–5A was diluted into Optima media (Invitrogen) and then mixed with Lipofectamine 2000 for 20 min. The mixture was added to the cells, incubated at 37°C for 3–5h, then the media was replaced with complete media plus serum.
Site-directed Mutagenesis of RNase L.
The RNase L point mutants were constructed using the QuikChange XL Site-Directed Mutagenesis Kit from Stratagene. Briefly, a full-length coding sequence DNA for human RNase L in pGEX-4T-3 vector (8)
was used as template for mutagenesis. The template plasmid was denatured and annealed with the two synthetic oligonucleotide primers containing the desired mutations. Using the nonstrand-displacing action of Pfu-Turbo DNA polymerase, we extended and incorporated the mutagenic primers, resulting in nicked circular strands. The methylated, nonmutated parental DNA template was digested with DpnI. The circular, nicked double-stranded DNA was transformed into XL10-Gold ultracompetent cells (Statagene). After transformation, the XL10-Gold ultracompetent cells repair the nicks in the mutated plasmid. All mutants were confirmed by DNA sequencing analysis.
Expression and Purification of GST-RNase L Fusion Proteins.
Briefly, the cDNAs for wild-type or mutant forms of RNase L in plasmid pGEX4-T-3 were transformed into Escherichia coli DH5
(8)
. The transformed bacteria were grown at 37°C to A595 = 0.5 before being induced with 0.1 mM isopropyl-1-thio-ß-D-galactpyranoside for 3 h. Harvested cell pellets were washed with PBS and resuspended in PBS-C (8)
. The suspended cells were sonicated on ice, and Triton X-100 was added to a final concentration of 1% (v/v). The supernatants were collected after centrifugation at 16,700 x g for 20 min at 4°C. Purification of fusion proteins were performed as described by the manufacturer of glutathione-Sepharose 4B (Pharmacia).
Activation of Purified Recombinant RNase L.
Substrate for in vitro RNase L assays was C7UUC12 (prepared on an ABI DNA synthesizer and purified as described above), labeled at its 3' terminus with [5'-32P]-pCp (3000 Ci/mmole; DuPont/New England Nuclear) with T4 RNA ligase (Life Technologies, Inc.; Ref. 18
). Briefly, 0.1 µg of purified GST-RNase L (wild type or R462Q) was incubated in the presence or absence of 0.1, 1, 10, and 100 nM 2–5A analogue on ice for 30 min. Reaction mixtures were incubated further with 80 nM C7UUC12-[32P]pCp for 30 min at 30°C. RNA was analyzed in sequencing gels to measure the extent of RNA degradation. The ratios of degraded RNA substrate to the intact RNA was quantitated in a phosphorimager.
Monitoring RNase L-mediated rRNA Cleavages in Intact Cells.
The cell-based RNase L assay was performed as described previously (1)
. Briefly, cells were transfected with 2–5A using Lipofectamine 2000 (Invitrogen) at the concentrations indicated in the figure legends. After a 3- to 5-h incubation, media were removed and cells were washed twice in 5 ml of PBS. Total RNA was isolated from transfected cells using the Trizol reagent according to manufacturer’s protocol and was quantitated by measuring absorbance at 260 nm. RNA (1 µg) was separated on RNA chips and analyzed with an Agilent Bioanalyzer 2100 (Agilent Technologies). The peak areas of 28S and 18S rRNA and their main cleavage products were measured using the Bio Sizing program (version A.02.01 S1232).
Cell Viability Assays.
The viability of cells was determined using the colorimetric CellTiter 96 Aqueous Cell Proliferation Assay, as described (19)
. Briefly, cells were seeded in 96-well culture plate (5 x 103 cells per well) and transfected with various forms of 2–5A at different concentrations. At 18 h after transfection, 50 µl of CellTiter 96 Aqueous reagent (40% v/v dilution in PBS) were added to each well. Plates were incubated at 37°C for 3 h, and absorbance was measured at 490 nm with a 96-well plate reader (model Spectra Max 340; Molecular Devices, Menlo Park, CA).
Western Blots.
Protein (100 µg) in cell extracts were separated in 10% polyacrylamide/SDS gels for detection of RNase L, PARP, or ß-actin or 12% polyacrylamide/SDS gels for caspase 3. The proteins were transferred to Immobilon-P membrane (Millipore), incubated with monoclonal antibody to human RNase L (20)
, or polyclonal antibody to human cleaved caspase 3 (Chemicon) or polyclonal antibody to PARP (Cell Signaling), or monoclonal antibody to ß-actin (Sigma) for 1 h. Membranes were washed with PBS with 1% Tween 20 and incubated with goat antimouse antibody or goat antirabbit antibody tagged with horseradish peroxidase (Life Technologies, Inc.) for 1 h. Proteins in the blots were detected by enhanced chemiluminesence (Amersham).
Binding of 2',5'-Oligoadenylates to RNase L.
A 32P-labeled and bromine-substituted 2–5A analogue, p(A2'p)2(br8A2'p)2A3'[32P]pCp (probe), was cross-linked to GST-RNase L (human; 0.4 µg) or to RNase L in crude cell extracts (200 µg) under UV light, as described (21)
. The RNase L fusion proteins or cell extracts were incubated with the probe, 105 cpm (specificity, 3000 Ci/mmol), in 50 µl of buffer on ice for 60 min. Samples were exposed to 308-nm light to induce covalent cross-linking to RNase L on ice for an additional 60 min. Protein separation was by electrophoresis in SDS/10% polyacrylamide gels, followed by autoradiography of the dried gels.
RNase L/RNase L Interaction Assays.
The RNase L dimerization assay was performed as described previously (8)
. Briefly, E. coli cell extracts (100 µg of protein) containing GST-RNase L or GST-RNase LR462Q were incubated with 60 µg of extract containing human recombinant (untagged) RNase L produced in insect cells (22)
in the presence or absence of different concentrations of 2–5A on ice for 1 h. BSA (250 µg) and 5 µl of 20% (v/v) glutathione sepharose 4B was added, and the mixtures were incubated with shaking at room temperature for 20 min, with gentle vortexing every 5 min, followed by washing three times with PBS-C (8)
. The immobilized proteins were eluted with SDS/gel sample buffer with boiling for 5 min, separated by electrophoresis in SDS/8% polyacrylamide gels, transferred to nitrocellulose membrane, and probed with monoclonal antibody to human RNase L (20)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Natural 5'-triphosphorylated 2–5A trimer and dimer (compounds 1 and 2) were synthesized enzymatically from ATP using 2–5A synthetase, whereas a 5'-monophosphoryl phosphodiester trimer (compound 3) and all other compounds were synthesized chemically (see "Materials and Methods"). Compounds 4 and 7 with 5'-thiophosphates and mixed isomer PS linkages had half-lives (t1/2) of >24 h at 37°C in human serum, compared with a t1/2 of only 1.2 h for a phosphodiester form of 2–5A (compound 3; Table 1
). Compounds 5–8 have 2'-terminal modifications designed for added stability, including a 2'-O-methyl group (compound 5), and 2' to 3' inverted deoxynucleotides (compounds 6–8). The PS derivatives of 2–5A (compounds 4, 5, 7, and 8) were shown to efficiently bind and activate RNase L in a cell-free system and in intact mouse fibroblast (L929) and/or human ovarian carcinoma (Hey1B) cells. As controls for nonspecific effects, compounds 2 and 6 lack the ability to activate RNase L because they contain only two instead of the requisite three consecutive 2',5'-linked adenylyl residues.
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. LNCaP cells are heterozygous for a deletion mutation (471AAAG), causing a frameshift and stop codon (12)
. In addition, the LNCaP cells are heterozygous for R462Q and D541E missense variants. RNase L in PC3 cells is homozygous for the wild-type accession sequence (NM021133), whereas in DU145 cells are heterozygous for a G296V missense variant of RNase L. Effects on cell viability of transfecting 2–5A compounds were determined by tetrazolium conversion (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium) assays. A PS 2–5A (compound 4) had a 10-fold enhanced anticellular effect on the DU145 cells compared with a natural phosphodiester (compound 1) (Fig. 2)
. The higher activity of compound 4 is likely caused by its enhanced biostability. In contrast, control diadenylate phosphodiester and phosphorothioate compounds that do not activate RNase L (compounds 2 and 6, respectively) had only slight effects on cell viability, suggesting that the anticellular effects of compounds 1 and 4 were because of RNase L activation. Indeed, transfections of compound 4 into DU145, PC3, and LNCaP cells produced characteristic RNase L-mediated cleavages in the 28S and 18S rRNA, as determined in RNA chips (Fig. 3)
. However, when treated with compound 4 at 1 µM, the LNCaP cells showed 2- and 1.4-fold less RNase L activity than the DU145 and PC3 cells. Similar results were obtained with natural 2–5A (data not shown).
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. Strong caspase 3 cleavage was also observed in the 2–5A-treated DU145 cells, whereas only slight caspase 3 cleavage was observed in the PC3 and LNCaP cells (Fig. 4B)
. Interestingly, RNase L levels as determined in immunoblots were similar in all three cell lines, despite the inactivating (frameshift) mutation in one allele of the LNCaP cells (Fig. 4C)
. Therefore, the RNASEL allele lacking the deletion is expressed to wild-type levels in LNCaP cells.
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. 2–5A treatment of the DU145, PC3, and LNCaP cells reduced cell viability to 2, 5, and 49% compared with the untreated cultures, as determined by tetrazolium conversion assays. In contrast, control compound 6 (10 µM) or mock treatments had relatively minor or no effects on DU145 and LNCaP cell viability. However, compound 6, at 10 µM, did reduce viability of the PC3 cells by 46% at 3 days. These findings demonstrate that the 2–5A analogues can induce apoptosis and/or significantly reduce proliferation of late-stage, metastatic prostate cancer cells when at least one active RNASEL allele is present.
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; Refs. 1
, 11
, and 14, 15, 16
). To determine the effects of the missense mutations, the variants were expressed individually in the mouse RNase L-/- rhabdomyosarcoma cell line JM03 (see "Materials and Methods"). Expressed levels of RNase L were measured in protein blots probed with monoclonal antibody against RNase L (Ref. 20
; Fig. 6A
). At 24 h after transfection of the RNase L cDNAs, cells were transfected with a relatively high concentration (50 µM) of a natural 2–5A mixture (see "Materials and Methods"). RNase L activity was measured from levels of intact 28S and 18S rRNA and specific rRNA cleavage products on RNA chips (1)
. Because the RNase L variants were expressed to different levels, rRNA cleavage levels were normalized to the level of RNase L that was expressed (Fig. 6A)
. Most of the variants had levels of RNase L activity that was similar to the wild-type enzyme. The Y529C variant was reduced by 35%, whereas the other variants had slightly less activity (
20%) than the wild-type enzyme. In contrast, the RNase LR462Q variant was reduced in activity by 3-fold compared with wild-type RNase L (Fig. 6B)
.
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. Because these assays were performed in vitro for relatively brief incubations, low concentrations (0.1 µM) of a phosphodiester 2–5A (compound 3) were used. RNase L cleaves the substrate on the 3' side of the UU dinucleotide sequence, leaving a 3'-phosphate. The R462Q variant had a 3-fold reduced rate of RNA cleavage compared with wild-type RNase L, consistent with our previous findings (15)
. To investigate the cause of the deficiency, 2–5A-binding activity was measured by covalent cross-linking of a radioactively labeled, bromine-substituted 2–5A analogue under UV light (21)
. However, the R462Q and wild-type enzymes had equivalent levels of 2–5A binding activity (Fig. 8)
.
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. Results indicate that the R462Q enzyme had a reduced capacity to dimerize into its activated state.
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. As a positive control, cleavage of both PARP and caspase 3 were observed in cells treated with tumor necrosis factor and actinomycin D (Fig. 10
, Lane 3). The 2–5A analogues did not induce cleavage of either PARP or caspase 3 in cells transfected with the empty vector (Fig. 10
, Lanes 4–8). In contrast, cleavage of both PARP and caspase 3 was observed in cells expressing wild-type RNase L (Fig. 10
, Lanes 10–13). PARP cleavage was reduced 2-fold in cells expressing RNase LR462Q, whereas caspase 3 cleavage was undetectable, despite expression of similar levels of RNase L and RNase LR462Q. These findings suggest that the association of RNase LR462Qwith prostate cancer risk (11
, 15) correlates with deficient enzymatic activity and consequently in a decreased ability to induce apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For example, the PS, biostable 2–5A analogue ps5'A(2'ps5'A)3 (compound 4) had an IC50 of 0.4 µM (concentration required to inhibit cell viability by 50%) in DU145 cells. In contrast, the IC50 of the natural phosphodiesterase 2–5A ppp5'A(2'p5'A)2 (compound 1) was 7 µM, or 17-fold higher. Despite observations that mouse RNase L requires a 5'-diphosphate on 2–5A, the monophosphorylated, PS analogues efficiently activated both mouse and human RNase L (Table 1
; Ref. 26
). Therefore, these compounds are predicted to be useful in rodent models of prostate cancer. The other advantages of these mixed isomer PS derivatives are a relatively simple mode of preparation compared with stereoisomers, high yields, and a straightforward method of purification.
Apoptosis of Prostate Cancer Cell Lines by 2–5A Activation of RNase L.
Results show that 2–5A activation of RNase L leads to RNA damage-mediated apoptosis in the metastatic prostate tumor cell lines DU145, PC3, and, to a lesser extent, LNCaP. Whereas PC3 and DU145 cells are homozygous for fully active forms of RNase L, the LNCaP cells have a deletion/frameshift mutation in one RNASEL allele. LNCaP cells express normal levels of RNase L as the result of allelic compensation. Nevertheless, LNCaP cells were less sensitive to 2–5A treatments than the other two cell lines. For instance, 2–5A produced 39, 27, and 19% rRNA breakdown in the DU145, PC3, and LNCaP cells, respectively (Fig. 3)
. Three days of 2–5A treatment reduced cell viability by 98% and 95% in the DU145 and PC3 cells, respectively, whereas about half of the LNCaP cells survived under identical treatments (Fig. 5)
. PARP cleavage in response to 2–5A was also greater in the DU145 and PC3 cells than in the LNCaP cells (Fig. 4A)
. 2–5A activation of RNase L has been shown to lead to release of cytochrome c from mitochondria and to caspase 3-dependent apoptosis (19)
. In these studies, caspase 3 cleavage in response to 2–5A treatment occurred to a greater extent in DU145 cells than in the PC3 and LNCaP cells (Fig. 4B)
. Therefore, PARP cleavage was a better indicator of cell death than caspase 3 cleavage. These results suggest that in the LNCaP cells a truncated RNase L produced from the mutant allele may act as an inhibitor of RNase L or that the LNCaP are less efficient in 2–5A uptake than the DU145 and PC3 cells. However, transfection of a fluorescein-tagged 2–5A into the three cell lines did not show a large difference in transfection efficiencies as measured by cytofluorimetry (data not shown). Therefore, it remains possible that the LNCaP cells produce a RNase L-truncated polypeptide that acts as dominant negative.
The R462Q Variant of RNase L Has a Reduced Capacity to Induce Apoptosis in Response to 2–5A.
Three inactivating mutations and an additional nine missense variants in RNASEL have been observed collectively in prostate cancer cases and/or in controls (Fig. 1)
(1
, 11
, 13, 14, 15, 16)
. All three inactivating mutations (M1I, E265X, and 157) and six missense variants (G59S, I97L, I220V, V247M, G296V, and S322F) map to the N-terminal half of RNase L that binds 2–5A, whereas the remaining three missense variants (R462Q, Y529C, and D541E) are in the protein kinase-like region. No mutations or variants have been observed in the RNase domain (Fig. 1)
. Among the missense mutants that have been examined in prostate cancer cases (S406F, D541E, I97L, and R462Q), only the R462Q variant has been shown to be associated with prostate cancer risk or aggressiveness (11
, 14
, 15)
. To determine the effect of the various missense mutations on enzyme activity, the wild-type and mutant forms of RNase L were compared after expression in mouse JM03 cells, isolated from a spontaneous rhabdomyosarcoma from RNase L-/- p53-/- double gene knockout mice. The R462Q variant showed the lowest levels of enzyme activity, approximately one-third of wild-type RNase L. The Y529C variant was reduced by 35%, whereas the other variants showed similar activity to the wild-type enzyme. These findings are consistent with genetic evidence implicating, thus far, only R462Q in prostate cancer risk.
A deficiency in RNase LR462Q was investigated further in this study using recombinant purified protein. Enzyme kinetics indicated a 3-fold reduction in catalytic rate compared with wild-type enzyme, consistent with our previous results (15)
. Whereas 2–5A binding activity was unaffected, the defect was related to a decreased capacity of the enzyme to dimerization into its active form. Previously, we found that another amino acid residue in the protein kinase-like domain of RNase L, K392, was required for dimerization (27)
. Our current results support a role for the protein kinase homology region in enzyme dimerization. The R462Q mutation reduced the ability of RNase L to cause apoptosis in response to activation by 2–5A as measured by cleavage of PARP and caspase 3 (Fig. 10)
. Therefore, the association of the R462Q mutant with prostate cancer risk correlates with a deficiency in apoptosis. These findings lend additional support to the notion that the tumor suppressor function of RNase L is related to its apoptotic activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USPHS Grants CA44059 and CA62220 from the Department of Health and Human Services. 
2 To whom requests for reprints should be addressed, at Department of Cancer Biology, NB40, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 445-9650; Fax: (216) 445-6269; E-mail: silverr{at}ccf.org
3 The abbreviations used are: HPLC, high-performance liquid chromatography; PARP, poly(ADP-ribose) polymerase; GST, glutathione S-transferase; PS, phosphorothioate.
4 J. Murakami and R. H. Silverman, unpublished observations.
Received 7/15/03. Accepted 9/ 2/03.
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REFERENCES |
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 A. Orr-Urtreger, A. Bar-Shira, D. Bercovich, N. Matarasso, U. Rozovsky, S. Rosner, S. Soloviov, G. Rennert, L. Kadouri, A. Hubert, et al. RNASEL Mutation Screening and Association Study in Ashkenazi and Non-Ashkenazi Prostate Cancer Patients. Cancer Epidemiol. Biomarkers Prev., March 1, 2006; 15(3): 474 - 479. [Abstract] [Full Text] [PDF]
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 J. Spence, B. M. Duggan, C. Eckhardt, M. McClelland, and D. Mercola Messenger RNAs under Differential Translational Control in Ki-ras-Transformed Cells Mol. Cancer Res., January 1, 2006; 4(1): 47 - 60. [Abstract] [Full Text] [PDF]
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 D. J. Bettoun, A. Scafonas, S. J. Rutledge, P. Hodor, O. Chen, C. Gambone, R. Vogel, S. McElwee-Witmer, C. Bai, L. Freedman, et al. Interaction between the Androgen Receptor and RNase L Mediates a Cross-talk between the Interferon and Androgen Signaling Pathways J. Biol. Chem., November 25, 2005; 280(47): 38898 - 38901. [Abstract] [Full Text] [PDF]
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K. Malathi, J. M. Paranjape, E. Bulanova, M. Shim, J. M. Guenther-Johnson, P. W. Faber, T. E. Eling, B. R. G. Williams, and R. H. Silverman A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L PNAS, October 11, 2005; 102(41): 14533 - 14538. [Abstract] [Full Text] [PDF]
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H. Rennert, C. M. Zeigler-Johnson, K. Addya, M. J. Finley, A. H. Walker, E. Spangler, D. G.B. Leonard, A. Wein, S. B. Malkowicz, and T. R. Rebbeck Association of Susceptibility Alleles in ELAC2/HPC2, RNASEL/HPC1, and MSR1 with Prostate Cancer Severity in European American and African American Men Cancer Epidemiol. Biomarkers Prev., April 1, 2005; 14(4): 949 - 957. [Abstract] [Full Text] [PDF]
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 K. Malathi, J. M. Paranjape, R. Ganapathi, and R. H. Silverman HPC1/RNASEL Mediates Apoptosis of Prostate Cancer Cells Treated with 2',5'-Oligoadenylates, Topoisomerase I Inhibitors, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cancer Res., December 15, 2004; 64(24): 9144 - 9151. [Abstract] [Full Text] [PDF]
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F. Wiklund, B.-A. Jonsson, A. J. Brookes, L. Stromqvist, J. Adolfsson, M. Emanuelsson, H.-O. Adami, K. Augustsson-Balter, and H. Gronberg Genetic Analysis of the RNASEL Gene in Hereditary, Familial, and Sporadic Prostate Cancer Clin. Cancer Res., November 1, 2004; 10(21): 7150 - 7156. [Abstract] [Full Text] [PDF]
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