Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC50 values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt. In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

BACKGROUND TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. METHODS Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. RESULTS TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). CONCLUSION TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Early life risk factors and puberty implications for cancer prevention

Earlier age at puberty is a known risk factor for breast cancer and suspected to influence prostate cancer; yet few studies have assessed early life risk factors for puberty. The overall objectives was to determine the relationship between birth-weight-for-gestational-age (BWGA), weight gain in infancy and pubertal status in girls and boys at 10.8 and 11.8 years and who were born of preeclamptic (PE) and normotensive (NT) mothers. Data for this study were collected from hospital and public health medical records and at a follow-up visit at 10.8 and 11.8 years for girls and boys, respectively. We used stratified analysis and multivariable logistic regression modeling to assess effect measure modifier and to determine the relationship between BWGA, weight gain in infancy and childhood and pubertal status, respectively. ^ There was no difference in the relationship between BWGA and pubertal status by maternal PE status for girls and boys; however, there was a non-significant increase in the odds of having been born small-for-gestational-age (SGA) in girls who were pubertal for breast or pubic hair Tanner stage 2+ compared to those who B1 or PH1. In contrast, boys who were pubertal for genital and pubic hair Tanner stage 2+ had lower odds of having been born SGA than those who were prepubertal for G1 or PH1. ^ In girls who were pubertal for breast development, the odds of having gained one additional unit SD for weight was highest between 3 to 6 months and 6-12 months for those who were B2+ vs. B1. For pubic hair development, weight gain between 6-12 months had the greatest effect for girls of PE mothers only. In boys, there were no statistically significant associations between weight gain and genital Tanner stage at any of the intervals; however, weight gain between 3-6 months did affect pubic hair tanner stage in boys of NT mothers. This study provide important evidence regarding the role of SGA and weight gain at specific age intervals on puberty; however, larger studies need to shed light on modifiable exposures for behavioral interventions in pregnancy, postpartum and in childhood.^

Bisphosphonates for metastatic bone disease in lung cancer

Bisphosphonates have proven effectiveness in preventing skeletal-related events (SREs) in advanced breast cancer, prostate cancer and multiple myeloma. The purpose of this study was to assess efficacy of bisphosphonates in preventing SREs, in controlling pain, and in increasing life expectancy in lung cancer patients with bone metastases.^ We performed an electronic search in MEDLINE, EMBASE, Web of Science, and Cochrane library databases up to April 4, 2010. Hand searching and searching in clinicaltrials.gov were also performed. Two independent reviewers selected all clinical trials that included lung cancer patients with bone metastases treated with bisphosphonates. We excluded articles that involved cancers other than lung, patients without bone metastasis and treatment other than bisphosphonates. Outcome questions answered were efficacy measured as overall pain control, overall improvement in survival and reduction in skeletal-related events or SREs (fracture, cord compression, radiation or surgery to the bone, hypercalcemia of malignancy). The quality of each study was evaluated using the Cochrane Back Review group questionnaire to assess risk of bias (0-worst to 11-best). Data extraction and quality assessments were independently performed by two assessors. Meta-analyses were performed where more than one study with similar outcomes were found.^ We identified eight trials that met our inclusion criteria. Three studies evaluated zoledronic acid, three pamidronate, three clodronate and two ibandronate. Two were placebocontrol trials while two had multi-group comparisons (radiotherapy, radionucleotides, and chemotherapy) and two had different bisphosphonate as active controls. Quality scores ranged from 1-4 out of 11 suggesting high risk of bias. Studies failed to report adequate explanation of randomization procedures, concealment of randomization and blinding. Metaanalysis showed that patients treated with zoledronic acid alone had lower rates of developing SREs compared to placebo at 21 months (RR=0.80, 95% CI=0.66-0.97, p=0.02). Meta-analyses also showed increased pain control when a bisphosphonate was added to the existing treatment modality like chemotherapy or radiation (RR=1.17, 95% CI=1.03-1.34, p=0.02). However, pain control was not statistically significantly different among various bisphosphonates when other treatment modalities were not present. Despite improvement in SRE and pain control, bisphosphonates failed to show improvement in overall survival (Difference in means=109.1 days, 95% CI= -51.52 – 269.71, p=0.183).^ Adding biphosphonates to standard care improved pain control and reduced SREs. Biphosphonates did not improve overall survival. Further larger studies with higher quality are required to stengthen the evidence.^ Keywords/MeSH terms Bisphosphonates/diphosphonates: generic, chemical and trade names.^

Participation of bcl -2 and bax in epidermal homeostasis and non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most frequently diagnosed form of cancer in United States. As in many other cancers, this slow growing malignancy manifests deregulated expression of apoptosis regulating proteins including bcl-2 family member proteins. To understand the role of apoptosis regulating protein in epidermal homeostasis and progression of NMSC, we investigated keratinocyte proliferation, differentiation and tumorigenesis in bcl-2 and bax null mice. The rate and the pattern of proliferation and spontaneous cell death were the same between the null and the control mice. Both bcl-2 and bax null epidermis showed decreased levels of cytokeratin 14 expression compared to the control littermates. Also, the gene knock out mice showed higher expression of cytokeratin 1 and loricrin in epidermis compared to the control mice. The apoptotic response to genotoxic agent, UV radiation (UVR), was assessed by counting sunburn cells. The bax null keratinocytes showed a resistance to apoptosis while bcl-2 null mice showed an increased susceptibility to cell death compared to the control mice. Moreover, we demonstrated an increase in tumor incidence in bax null mice compared to control littermates in the in vivo chemical carcinogenesis study. Next, we examined the tumor suppressor role of bax protein in NMSC by studying its participation in repair of UVR-mediated DNA lesions. In UVR treated primary keratinocytes from bax deficient mice, the level of CPD remaining was twice that of control cells at 48 hours. Similar results were obtained using embryonic fibroblasts from bax null and bax +/+ embryos, and also with a bax deficient prostate cancer cell line in which bax expression had been restored. However, the repair rate of 6-4 PP was unaffected by the absence of bax protein in all three of above mentioned cell types. In conclusion, bax protein may have a dual function in its role as tumor suppressor in NMSC. Bax may directly or indirectly facilitate DNA repair, or programmed cell death if DNA damage is too severe, thus, in either function, preserving genomic integrity following a genotoxic event. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral γ34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 γ34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a γ34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the γ34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers.

Human prostate tumor growth in athymic mice: inhibition by androgens and stimulation by finasteride.

When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for > 100 passages, the cells, now called LNCaP 104-R2, are proliferatively repressed by low concentrations of androgens. LNCaP 104-R2 cells formed tumors in castrated male athymic nude mice. Testosterone propionate (TP) treatment prevented LNCaP 104-R2 tumor growth and caused regression of established tumors in these mice. Such a tumor-suppressive effect was not observed with tumors derived from LNCaP 104-S cells or androgen receptor-negative human prostate cancer PC-3 cells. 5 alpha-Dihydrotestosterone, but not 5 beta-dihydrotestosterone, 17 beta-estradiol, or medroxyprogesterone acetate, also inhibited LNCaP 104-R2 tumor growth. Removal of TP or implantation of finasteride, a 5 alpha-reductase inhibitor, in nude mice bearing TP implants resulted in the regrowth of LNCaP 104-R2 tumors. Within 1 week after TP implantation, LNCaP 104-R2 tumors exhibited massive necrosis with severe hemorrhage. Three weeks later, these tumors showed fibrosis with infiltration of chronic inflammatory cells and scattered carcinoma cells exhibiting degeneration. TP treatment of mice with LNCaP 104-R2 tumors reduced tumor androgen receptor and c-myc mRNA levels but increased prostate-specific antigen in serum- and prostate-specific antigen mRNA in tumors. Although androgen ablation has been the standard treatment for metastatic prostate cancer for > 50 years, our study shows that androgen supplementation therapy may be beneficial for treatment of certain types of human prostate cancer and that the use of 5 alpha-reductase inhibitors, such as finasteride or anti-androgens, in the general treatment of metastatic prostate cancer may require careful assessment.

Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.

Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.