Differential regulation of PTEN expression by androgen receptor in prostate and breast cancers.

Prostate cancer and breast cancer are the most common malignancies in the western world. Androgen receptor (AR) and PTEN both have been well documented to have important roles in prostate carcinogenesis. In contrast, AR and PTEN in breast carcinogenesis have not been well studied. Furthermore, the crosstalk and connection between those two pathways remain unclear. Increased AR expression in prostate cancers, combined with decreased PTEN expression, portends a poor clinical outcome. Paradoxically, both high AR and high PTEN levels, detected by immunohistochemistry, in primary breast carcinomas have been associated with better disease-free survival. Here, we performed in silico analysis of publicly available microarray data sets from prostate or breast carcinomas. We found an inverse correlation between AR and PTEN transcript expression in prostate cancer tissues in contrast to the positive correlation in breast cancer. These data led us to hypothesize that AR may directly affect PTEN transcriptional regulation in prostate and breast cancer cells. Here, we show for the first time that AR inhibits PTEN transcription in prostate cancer cells, whereas AR upregulates PTEN transcription in breast cancer cells, which mechanistically explains both the immunohistochemical PTEN-AR expressional data noted in clinical trials and in our in silico analysis of the transcriptomes of breast and prostate cancers. In addition, we have fine-mapped the AR-binding motif within the PTEN promoter. Here we show that, in patients with Cowden syndrome, an inherited cancer syndrome caused by germline mutations scattered throughout PTEN, point variants affecting the 3' end of the AR-binding motif result in abrogation of androgen-mediated transcriptional regulation of PTEN expression. We may speculate that the differential AR effect on PTEN may begin to explain organ-specific and perhaps sex-specific neoplasia predisposition in Cowden syndrome, as well as why only a fraction of women with germline PTEN mutations develop breast cancer, depending on the androgen steroid milieu and levels.

The Flvr-encoded murine oligoadenylate synthetase 1b (Oas1b) suppresses 2-5A synthesis in intact cells.

Resistance to flavivirus-induced disease in mice is conferred by the autosomal gene Flv, identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Resistant mice express a full-length Oas1b protein while susceptible mice express the truncated Oas1btr. In this study, Oas1b was shown to be an inactive synthetase. Although the Oas/RNase L pathway was previously shown to have an antiviral role during flavivirus infections, Oas1b protein inhibited Oas1a in vitro synthetase activity in a dose-dependent manner and reduced 2-5A production in vivo in response to poly(I:C). These findings suggest that negative regulation of 2-5A by inactive Oas1 proteins may fine tune the RNase L response that if not tightly controlled could cause significant damage in cells. The results also indicate that flavivirus resistance conferred by Oas1b is not mediated by 2-5A. Instead, Oas1b inhibits flavivirus replication by an alternative mechanism that overrides the proviral effect of reducing 2-5A accumulation and RNase L activation.

RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth.

Ribonuclease L (RNase L) is an intracellular enzyme that is vital in innate immunity, but also is a tumor suppressor candidate. Here, we show that overexpression of RNase L decreases cellular growth and downmodulates the RNA-binding protein, HuR, a regulator of cell-cycle progression and tumorigenesis. The effect is temporal, occurring in specific cell-cycle phases and correlated with the cytoplasmic localization of RNase L. Both cellular growth and HuR were increased in RNASEL-null mouse fibroblast lines when compared to wild-type cells. Moreover, the stability of HuR mRNA was enhanced in RNASEL-null cells. The HuR 3' untranslated region (UTR), which harbors U-rich and adenylate-uridylate-rich elements, was potently responsive to RNase L when compared to control 3' UTR. Our results may offer a new explanation to the tumor suppressor function of RNase L.

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis.

IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF-alpha related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-alpha, IFN-beta and IFN-gamma induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.

Monitoring activation of ribonuclease L by 2',5'-oligoadenylates using purified recombinant enzyme and intact malignant glioma cells.

A wide range of methods have been developed to study RNase L activation in cell-free systems and in intact cells. Many of the original methods were developed in the laboratory of I. Kerr in the early 1980s (e.g., see Knight et al.). Additional methods described in this article were developed or adapted from research in other fields after the cloning of RNase L and the appreciation of its role in apoptosis. These methods provide the basic techniques needed to induce RNase L activation in vitro and to measure some of its biological effects in living mammalian cells.

Chemistry and biochemistry of 2',5'-oligoadenylate-based antisense strategy.

This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described.

Antisense cancer therapy: the state of the science.

Over the last few years, antisense technology has emerged as an exciting and promising strategy in the fight against cancer. The antisense concept is to selectively bind short, modified DNA or RNA molecules to messenger RNA in cells and prevent the synthesis of the encoded protein. As anticancer agents, these molecules can be targeted against a myriad of genes involved in cell transformation, cell survival, metastasis, and angiogenesis. Indeed, the list of possible antisense targets increases as the knowledge of the genetic basis of oncogenesis expands. One aim of this review is to focus on those antisense cancer drugs that have entered human clinical trials. At least four of these compounds are currently in phase II trials, including those targeting protein kinase C-alpha, bcl-2, c-raf, and the R1-alpha subunit of protein kinase A. A new development in antisense chemistry (peptide nucleic acids) is discussed, along with alternative antisense-related strategies (ribozymes and 2-5A-antisense) designed to overcome some of the challenges of this already encouraging technology.

Arc-scanning very high-frequency digital ultrasound for 3D pachymetric mapping of the corneal epithelium and stroma in laser in situ keratomileusis.

PURPOSE: To test and demonstrate measurement precision, imaging resolution, 3D thickness mapping, and clinical utility of a new prototype 3D very high-frequency (VHF) (50 MHz) digital ultrasound scanning system for corneal epithelium, flap, and residual stromal thickness after laser in situ keratomileusis (LASIK). METHODS: VHF ultrasonic 3D data was acquired by arc-motion, meridional scanning within a 10-mm zone. Digital signal processing techniques provided high-resolution B-scan imaging, and I-scan traces for high-precision pachymetry in 4 eyes. Thickness maps of individual corneal layers were constructed. Reproducibility of epithelial, flap, and full corneal pachymetry was assessed for single-point and 3D thickness mapping by repeated measures. Thickness mapping of the epithelium, stroma, flap, and full cornea were determined before and after LASIK. Preoperative to postoperative difference maps for epithelium, flap, and stroma were produced to demonstrate anatomical changes in the thickness profile of each layer. RESULTS: Surface localization precision was 0.87 microm. Central reproducibility for single-point pachymetry of epithelium was 0.61 microm; flap, 1.14 microm; and full cornea, 0.74 microm. Reproducibility for central pachymetry on 3D thickness mapping was 0.5 microm for epithelium and 1.5-microm for full cornea. B-scans and 3D thickness maps after LASIK demonstrated resolution of epithelial, stromal component of the flap, and residual stromal layers. Large epithelial profile changes were demonstrated after LASIK. Topographic variability of flap thickness and residual stromal thickness were significant. CONCLUSIONS: VHF digital ultrasound arc-B scanning provides high-resolution imaging and high-precision three-dimensional thickness mapping of corneal layers, enabling accurate anatomical evaluation of the changes induced in the cornea by LASIK.

2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells.

OBJECTIVE: RNase L is converted to an active form upon binding short 2',5'-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase-an RNA-protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B. METHODS: Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained. RESULTS: Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death. CONCLUSIONS: The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.

Caspase-dependent apoptosis by 2',5'-oligoadenylate activation of RNase L is enhanced by IFN-beta.

The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biologic action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. RNase L is also implicated in mediating apoptosis in response to both viral and nonviral inducers. To study the cellular effects of RNase L activation directly, 2-5A was transfected into the human ovarian cancer cell line, Hey1B. Activation of RNase L by 2-5A resulted in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays. In contrast, the dimeric form of 2-5A, ppA2'p5'A, neither activated RNase L nor caused apoptosis. Treatment with IFN-beta prior to 2-5A transfection enhanced cellular RNase L levels (< or = 2.2-fold) and increased the proportion of cells undergoing apoptosis (by < or =40%). However, rRNA cleavages after 2-5A transfections were not enhanced by IFN-beta pretreatments, indicating that basal levels of RNase L were sufficient for this activity. Apoptosis in response to RNase L activation was accompanied by cytochrome c release from mitochondria. Induction of apoptosis by either 2-5A alone or by the combination of 2-5A and IFN-beta was effectively blocked with either the pancaspase inhibitor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-fmk. Therefore, activation of RNase L by 2-5A leads to cytochrome c release into the cytoplasm and then to caspase activation and apoptosis. These results suggest potential uses for 2-5A in augmenting the anticancer activities of IFN.

Transcriptional control of the human plasma membrane phospholipid scramblase 1 gene is mediated by interferon-alpha.

Interferons (IFNs) mediate their diverse biologic activities through induction of the expression of multiple genes. Whereas the mode of action of certain of these IFN-regulated genes has been well characterized, most of the molecular and cellular events underlying the constellation of biologic responses to the IFNs remain unresolved. This study showed that the newly identified PLSCR1 gene for phospholipid scramblase, previously implicated in remodeling of plasma membrane phospholipids, is regulated at the transcriptional level by IFN-alpha. Analysis of 5' flanking genomic sequence in reporter constructs showed that transcriptional control of PLSCR1 was entirely regulated by a single IFN-stimulated response element located in the first exon. A similar induction of PLSCR1 by IFN-alpha2a was also observed in a variety of other human tumor cell lines as well as in human umbilical vein endothelial cells. In these cell lines, the marked IFN-alpha2a-induced increase in PLSCR1 protein expression, ranging as high as 10-fold above basal levels, was not accompanied by increased cell surface exposure of phosphatidylserine, suggesting that remodeling of the cell surface requires both exposure to IFN and a second yet-to-be identified event to stimulate plasma membrane phospholipid scramblase activity and to mobilize phosphatidylserine to the cell surface. (Blood. 2000;95:2593-2599)

Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

The antiviral enzymes PKR and RNase L suppress gene expression from viral and non-viral based vectors.

Expression of transfected genes is shown to be suppressed by two intracellular enzymes, RNase L and protein kinase PKR, which function in interferon-treated cells to restrict viral replication. RNase L(-/-) or PKR(-/-) murine embryonic fibroblasts produced enhanced levels of protein from transfected genes compared with wild-type cells. Increased expression of exogenous genes in RNase L(-/-) cells correlated with elevated levels of mRNA and thus appeared to be due to enhanced mRNA stability. Plasmid encoding adenovirus VA RNAs was able to further enhance accumulation of the exogenous gene transcript and protein, even in cells lacking PKR. In contrast to the increased expression of transfected genes in cells lacking RNase L or PKR, expression of endogenous host genes was unaffected by the absence of these enzymes. In addition, a dominant-negative PKR mutant improved expression from a conventional plasmid vector and from a Semliki Forest virus derived, self-replicating vector. These results indicate that viral infections and transfections produce similar stress responses in mammalian cells and suggest strategies for selectively increasing expression of exogenous genes.

High-resolution ultrasonic imaging of blood flow in the anterior segment of the eye.

PURPOSE: To develop a noninvasive technique to visualize and measure blood flow in the iris and ciliary body. METHODS: Echo data from 50-MHz ultrasound scans of the iris and ciliary body of rabbits were digitized using a new "swept scan" modality. The method makes use of spatial oversampling to identify regions with scatterers whose range changes with time. The data allowed construction of high-resolution B-mode images with embedded flow information. Pulsatility over the cardiac cycle was evaluated by sending a series of pulses along a single line of sight containing a vessel of interest. Local blood flow and changes over the cardiac cycle before and after application of atropine were quantified. RESULTS: Flow was identified in the radial vessels and major arterial circle of the iris. Vessels with lumens as small as 40 microm in diameter and flow velocities as low as 0.6 mm/sec were measured. Change in blood velocity over the cardiac cycle was determined to be approximately 27%. Peak systolic velocity after administration of topical atropine increased by 72%. CONCLUSIONS: This technique allowed visualization of flow using the same type of very-high-frequency transducer now widely used for imaging the anterior segment. The technique can also be used at lower frequencies for more posterior tissues with similar improvement of resolution over Doppler. The ability to examine flow in the anterior segment of the eye offers a new tool for study of glaucoma, hypotony, tumors, and other disorders.

A comparison of ultrasonic beams for thermal treatment of ocular tumors.

OBJECTIVE: This study examined the relative merits of different ultrasonic beams and exposure modalities for treating ocular melanomas. METHODS: Simulations were conducted to evaluate temperature patterns and lesion shapes induced by intense-ultrasound treatment of ocular tumors. In-vitro insonification experiments were conducted in bovine lenses. RESULTS: Simulated hyperthermia exposures did not effectively treat tumor margins because of thermal conduction into nearby fluid-like media. Standard high-intensity focused beams produced narrow lesions during 2-s exposures. A high-intensity, multi-lobed beam, produced by a transducer with strip electrodes, generated asymmetric lesions with a single large dimension; this lesion shape could expedite the production of lesion matrices within large tumors. In-vitro cataract shapes were consistent with simulation results for focused high-intensity beams. CONCLUSIONS: Thermal conduction and perfusion can cause underheating of tumor margins during hyperthermia unless special beam designs are used. The strip-electrode transducer configuration promises to expedite treatment of extended tumor volumes.

Very high-frequency ultrasound corneal analysis identifies anatomic correlates of optical complications of lamellar refractive surgery: anatomic diagnosis in lamellar surgery.

OBJECTIVE: To examine the utility of very high-frequency (VHF) ultrasound scanning in determining the anatomic changes and correlates of optical complications in lamellar refractive surgery. STUDY DESIGN: Case series. PARTICIPANTS: Cases analyzed included marked asymmetric astigmatism postautomated lamellar keratoplasty (ALK), image ghosting despite normal videokeratography post-ALK, uncomplicated myopic laser in situ keratomileusis (LASIK), and hyperopic LASIK with regression. METHODS: A prototype VHF ultrasound scanner (50 MHz) was used to obtain sequences of parallel B-scans of the cornea. Digital signal processing techniques were used to measure epithelial, stromal, and flap thickness values in a grid encompassing the central 4 to 5 mm of the cornea, enabling pachymetric mapping of each layer with 2-micron precision. MAIN OUTCOME MEASURE: The appearance of the corneas in VHF ultrasound images and thickness values of individual corneal layers determined from VHF ultrasound data. RESULTS: VHF ultrasound resolved the epithelial, stromal cap, or flap and residual stromal layers 1 year after lamellar surgery. Asymmetric stromal tissue removal was differentiated from stromal cap irregularity. Epithelium acted to compensate for asymmetry of the stromal surface about the visual axis and for localized surface irregularities. Irregularities in the epithelial-stromal interface accounted for image ghosting present despite apparently normal videokeratography. Epithelial thickening was shown after uncomplicated myopic LASIK. Hyperopic LASIK demonstrated relative epithelial thickening localized to the region of ablation accounting for refractive regression. CONCLUSIONS: VHF ultrasound shows promise as a sensitive method of determining the anatomic correlates of optical complications in lamellar refractive surgery.

Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L.

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.

2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells.

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.