The androgen receptor is a negative regulator of eIF4E phosphorylation at S209: implications for the use of mTOR inhibitors in advanced prostate cancer.

The antiandrogen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration-resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pretreated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eukaryotic initiation factor 4E (eIF4E) phosphorylation, while the use of antiandrogens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. Small interfering RNA-mediated knockdown (k/d) of eIF4E-sensitized CRPC cells to RAD001+bicalutamide, whereas eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of mitogen-activated protein kinase-interacting serine-threonine kinases MAP kinase-interacting kinase 1 (Mnk1/2), the eIF4E upstream kinase) or inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2), the upstream kinase-regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation, whereas its inhibition restored cap-dependent translation, which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.

Toward enhanced pharmacovigilance using patient-generated data on the internet.

The promise of augmenting pharmacovigilance with patient-generated data drawn from the Internet was called out by a scientific committee charged with conducting a review of the current and planned pharmacovigilance practices of the US Food and Drug Administration (FDA). To this end, we present a study on harnessing behavioral data drawn from Internet search logs to detect adverse drug reactions (ADRs). By analyzing search queries collected from 80 million consenting users and by using a widely recognized benchmark of ADRs, we found that the performance of ADR detection via search logs is comparable and complementary to detection based on the FDA's adverse event reporting system (AERS). We show that by jointly leveraging data from the AERS and search logs, the accuracy of ADR detection can be improved by 19% relative to the use of each data source independently. The results suggest that leveraging nontraditional sources such as online search logs could supplement existing pharmacovigilance approaches.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model.

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Pyrocarbon metacarpophalangeal joint replacement in primary osteoarthritis.

The purpose of this retrospective cohort study was to evaluate the outcomes of 18 primary pyrocarbon metacarpophalangeal joint replacements in 10 patients, performed for primary osteoarthritis. The mean age at operation was 66 years and mean follow-up was 58.6 months. The arc of motion improved from a mean of 30° to 40° and the mean QuickDASH score improved from 35 to 17. All except one patient were satisfied with their outcomes. Radiographically, there has been no evidence of dislocation or overt loosening, although there has been subsidence of some components up to 5 mm. One index finger implant was revised to a silastic implant for perceived alteration of precision pinch. Other complications included one intra-operative fracture that united and an asymptomatic stem fracture of one proximal phalangeal component. We continue to use the implant and aim to review our experience in a further 5 years.

miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT.

Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3' untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.

Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells.

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

The effect of sirolimus on prostate-specific antigen (PSA) levels in male renal transplant recipients without prostate cancer.

In kidney recipients, the immunosuppressant sirolimus has been associated with a decreased incidence of de novo posttransplant malignancies (including prostate cancer). But the effect of sirolimus on the prostate-specific antigen (PSA) blood level, an important prostate cancer screening tool, remains unknown. We studied male kidney recipients >50 years old (transplanted from January 1994 to December 2006) without clinical evidence for prostate cancer. Pre- and posttransplant PSA levels were analyzed for 97 recipients (n = 19 on sirolimus, n = 78 on tacrolimus [control group]). Pretransplant PSA was similar for sirolimus versus tacrolimus recipients (mean, 1.8 versus 1.7 ng/mL, p = 0.89), but posttransplant PSA was significantly lower for recipients on sirolimus (mean, 0.9 versus 1.9 ng/mL, respectively, p < 0.001). The mean difference between pretransplant and posttransplant PSA was -0.9 ng/mL (50.0%, p = 0.006) for the sirolimus group versus +0.2 ng/mL (+11.8%, p = 0.24) for the tacrolimus group. By multivariate analysis, only pretransplant PSA and immunosuppression with sirolimus independently impacted posttransplant PSA. Our data strongly suggest that sirolimus is associated with a significant PSA decrease in kidney recipients. Future studies must investigate the clinical implications of our findings for the use of PSA for prostate cancer screening in male kidney recipients on sirolimus.

Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival.

The mTOR (mammalian target of rapamycin) inhibitor rapamycin caused growth arrest in both androgen-dependent and androgen-independent prostate cancer cells; however, long-term treatment induced resistance to the drug. The aim of this study was to investigate methods that can overcome this resistance. Here, we show that rapamycin treatment stimulated androgen receptor (AR) transcriptional activity, whereas suppression of AR activity with the antiandrogen bicalutamide sensitized androgen-dependent, as well as AR-sensitive androgen-independent prostate cancer cells, to growth inhibition by rapamycin. Further, the combination of rapamycin and bicalutamide, but not the individual drugs, induced significant levels of apoptosis in prostate cancer cells. The net effect of rapamycin is determined by its individual effects on the mTOR complexes mTORC1 (mTOR/raptor/GbetaL) and mTORC2 (mTOR/rictor/sin1/GbetaL). Inhibition of both mTORC1 and mTORC2 by rapamycin-induced apoptosis, whereas rapamycin-stimulation of AR transcriptional activity resulted from the inhibition of mTORC1, but not mTORC2. The effect of rapamycin on AR transcriptional activity was mediated by the phosphorylation of the serine/threonine kinase Akt, which also partially mediated apoptosis induced by rapamycin and bicalutamide. These results indicate the presence of two parallel cell-survival pathways in prostate cancer cells: a strong Akt-independent, but rapamycin-sensitive pathway downstream of mTORC1, and an AR-dependent pathway downstream of mTORC2 and Akt, that is stimulated by mTORC1 inhibition. Thus, the combination of rapamycin and bicalutamide induce apoptosis in prostate cancer cells by simultaneously inhibiting both pathways and hence would be of therapeutic value in prostate cancer treatment.

Inappropriate activation of androgen receptor by relaxin via beta-catenin pathway.

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) with subsequent cytoplasmic accumulation of beta-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized beta-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/beta-catenin complex binds to the proximal region of the prostate-specific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3beta and translocation of beta-catenin/AR into the nucleus. Knockdown of beta-catenin levels using a beta-catenin-specific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Inhibition of Akt pathways in the treatment of prostate cancer.

Akt is a serine/threonine kinase mediating multiple intracellular pathways involved in prostate cancer (CaP) biology. Increased understanding of the molecular mechanisms of Akt activation and signaling have led to the development of an increasing number of Akt inhibitors. These biologic agents demonstrate activity against a wide range of cancers in preclinical studies. Clinical studies of Akt inhibition in CaP are in progress, including agents such as celecoxib, perifosine and genistein. How best to integrate Akt inhibitors with standard CaP therapy or select patients most likely to benefit is the subject of ongoing research.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7.

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53(R273H)) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53(R273H)-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53(R273H) binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53(R273H)-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

Abnormalities of apoptotic and cell cycle regulatory proteins in distinct histopathologic components of benign prostatic hyperplasia.

INTRODUCTION: Benign prostatic hyperplasia (BPH) is a slowly progressive abnormal glandular enlargement with heterogeneous morphology. Disruption of apoptotic pathways has been suggested as an important regulatory mechanism in this common and significantly morbid disease. METHODS: Prostatic tissue from 20 patients with BPH and no prior or subsequent prostatic carcinoma was obtained by transurethral prostatectomy (TURP) at the University of California Davis. Apoptotic regulatory proteins: BCL2, BAX and p27 were analyzed by immunohistochemistry and evaluated for expression in four distinct histologic patterns: hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands. RESULTS: Particularly striking was the decreased expression of BAX and an abnormal BCL2 : BAX ratio within all nodules relative to expression in other epithelial patterns. p27 expression was decreased in 35% of the hyperplastic epithelial areas and 10% of the nodules. DISCUSSION: Overall, abnormal expression of BCL2, BAX and/or p27 was identified in the hyperplastic epithelium of 19 (90%) of specimens and all 12 (100%) of the hyperplastic nodules. The high frequency of abnormalities in apoptosis regulatory genes, suggests that alteration of apoptotic pathways is important for the development of this condition.

Can single dose preoperative intrathecal morphine sulfate provide cost-effective postoperative analgesia and patient satisfaction during radical prostatectomy in the current era of cost containment?

We retrospectively analyzed the analgesic efficacy and surgical outcomes of a single preoperative intrathecal long-acting morphine sulfate injection (0.25-0.5 mg) and postoperative intravenous (i.v.) ketorolac in 62 patients who underwent radical retropubic prostatectomy (RRP). Total postoperative analgesic requirement was documented along with assessment of length of hospital stay, pain control and time for resumption of normal activity. Postoperatively, 45% of patients required only nonsteroidal agents (ketorolac), whereas 55% needed a mean of 13.3 mg of supplemental i.v. morphine sulfate. Mean hospital stay was 2.3+/-0.3 days. Eighty-two per cent of patients felt the length of hospital stay adequate. Ninety-seven per cent of patients were satisfied with anesthesia selected and 95% of patients considered pain control on postoperative days 1 and 2 as effective. All patients resumed to full physical activity by 5.3+/-0.4 weeks after surgery. We conclude that a single preoperative injection of intrathecal morphine sulfate combined with i.v. ketorolac postoperatively results in effective analgesia, diminished supplemental narcotic requirement and high patient satisfaction during radical retropubic prostatectomy.

Phylogenetic relationships within the eared seals (Otariidae: Carnivora): implications for the historical biogeography of the family.

Phylogenetic relationships within the family Otariidae were investigated using two regions of the mitochondrial genome. A 360-bp region of the cytochrome b gene was employed for the primary phylogenetic analysis, while a 356-bp segment of the control region was used to enhance resolution of the terminal nodes. Traditional classification of the family into the subfamilies Arctocephalinae (fur seals) and Otariinae (sea lions) is not supported, with the fur seal Callorhinus ursinus having a basal relationship relative to the rest of the family. This is consistent with the fossil record which suggests that this genus diverged from the line leading to the remaining fur seals and sea lions about 6 million years ago (mya). There is also little evidence to support or refute the monophyly of sea lions. Four sea lion clades and five fur seal clades were observed, but relationships among these clades are unclear. Similar genetic divergences between the sea lion clades (D(a) = 0.054-0.078), as well as between the major Arctocephalus fur seal clades (D(a) = 0.040-0.069) suggest that these groups underwent periods of rapid radiation at about the time they diverged from each other. Rapid radiations of this type make the resolution of relationships between the resulting species difficult and indicate the requirement for additional molecular data from both nuclear and mitochondrial genes. The phylogenetic relationships within the family and the genetic distances among some taxa highlight inconsistencies in the current taxonomic classification of the family.

Differences in gene expression in muscle-invasive bladder cancer: a comparison of Italian and American patients.

OBJECTIVE: To seek differences in gene expression in the primary muscle-invasive bladder cancers of two cohorts of patients having different survival rates. An Italian group treated by transurethral resection of the bladder tumor (TURBT) and neo-adjuvant chemotherapy using methotrexate, vinblastine, adriamycin and cisplatin (M-VAC) followed by TURBT, partial cystectomy or radical cystectomy (75% 3-year survival) was compared to an American cohort treated by radical cystectomy (51% 3-year survival). METHODS: Immunohistochemistry was used to examine the protein expression levels of three genes that act at the G1/S cell cycle checkpoint, p53, p21/waf-1/cip1 (a downstream effector gene in the p53 pathway) and Rb, plus a major inhibitor of apoptosis, Bcl-2. RESULTS: For the bladder cancers of the Italian patient cohort, there was a significantly higher rate of p53 immunopositivity (93 vs. 63%, p = 0.002) and a significantly lower rate of Rb loss (25 vs. 54%, p = 0.009). In bivariate analysis, 72% of Italian tumors were immunopositive for both p53 and p21 (p53+/p21+) vs. 49% for the American tumors. The subset of Italian patients with p53+/p21+ tumors were more frequently disease-free (stage pT0) following chemotherapy and were less likely to fail therapy than those with p53+/p21- tumors (p = 0.0357). Loss of Rb staining was associated with a decreased 5-year survival in the Italian, but not in the American patients. CONCLUSIONS: (1) Significant differences in the expression of the p53, p21 and Rb genes were found between the 2 groups of patients. (2) Italian patients with p53+/p21+ tumors had significantly lower recurrence rates after TURBT and chemotherapy than those having p53+/p21- tumors. (3) Absence of p21 immunopositivity in the Italian tumors may identify alterations in the p53 pathway that predict poor outcome.

BCL2 antisense transcripts decrease intracellular Bcl2 expression and sensitize LNCaP prostate cancer cells to apoptosis-inducing agents.

Prostate cancer (CaP) is the most commonly diagnosed cancer of aging men and the second leading cause of male cancer death in the United States. At present, no effective therapy is available for treating hormone independent CaP. Since Bcl2 is believed to play a role in protecting CaP cells from apoptosis, we investigated the effects of down-regulating Bcl2 expression on CaP cells. Genetically engineered LNCaP sublines were established by stably transfecting LNCaP cells with BCL2 antisense (BCL2-AS) transcript-expressing plasmids. Western blotting analysis showed that intracellular Bcl2 protein was decreased by 50-60% in BCL2-AS-transfected LNCaP cells. Expression of the antisense transcripts resulted in 50% growth inhibition of LNCaP cells in response to androgen withdrawal and markedly sensitized these cells to Adriamycin-induced apoptosis. These results suggest that down-regulation of Bcl2 protein using BCL2-AS transcripts could be exploited for improved treatment of advanced CaP.