Design, syntheses, and characterization of pharmacophore based chemokine receptor CCR5 antagonists as anti prostate cancer agents.

Accumulating evidence has shown multiple roles that chemokine receptor CCR5 may play to promote the progression of several types of cancer. The mechanism of such promotion is believed to involve chronic inflammation that creates a microenvironment which enhances tumor survival. Therefore, blocking CCR5 function with an antagonist may provide a novel treatment of cancers such as prostate cancer. Currently, several CCR5 antagonists are available, but all have been optimized for their inhibitory activity on HIV-1 cellular membrane invasion process rather than inhibition on cytoplasmic signaling pathways. Thus, there is need to develop CCR5 antagonists focusing on blockage of CCR5 downstream signaling and inhibition of CCR5 related prostate cancer proliferation and progression. In this report, a pharmacophore analysis was conducted based on docking studies of several known CCR5 antagonists in a CCR5 homology model. A unique structural skeleton for CCR5 antagonist was constructed and functionalized, resulting in a new series of small molecules to be synthesized and characterized. A combination of CCR5 calcium flux inhibition, anti prostate cancer cell proliferation, basal cytotoxicity, and in vivo animal model studies were applied to screen the newly synthesized compounds. Results from this study provided a potential lead compound for future CCR5 antagonist development focusing on prostate cancer therapy.

Structure activity relationship studies of natural product chemokine receptor CCR5 antagonist anibamine toward the development of novel anti prostate cancer agents.

Recent studies have indicated that the CCR5 chemokine receptor may be a potential target for treating prostate cancer. Thus, development of CCR5 antagonists may provide novel prostate cancer therapy. Anibamine, a novel pyridine quaternary alkaloid isolated from Aniba sp., was found to effectively compete with (125)I-gp120 in binding to the chemokine receptor CCR5, with an IC(50) = 1 µM. Anibamine is the first natural product reported as a CCR5 antagonist, and thus provides a novel structural skeleton unique from other lead compounds that have generally been identified from high-throughput screening efforts. In order to refine the lead compound's structure and improve the therapeutic index of anibamine derivatives as potential anti prostate cancer agents, the approach of "deconstruction-reconstruction-elaboration" was applied in the structure-activity relationship studies of this work. Here, we report the design, syntheses and anti prostate cancer activities of anibamine and 17 analogues. The results from the in vitro and in vivo studies described here show that this class of compounds has potential to provide novel leads as anti prostate cancer agents.

The natural product CCR5 antagonist anibamine and its analogs as anti-prostate cancer agents.

Prostate cancer is a leading cause of death among males in the United States. As the chemokine receptor CCR5 is over-expressed in more aggressive forms of prostate cancer, and is also a critical receptor in inflammation, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. Anibamine, a natural product CCR5 antagonist, provides a unique molecular scaffold for the generation of novel analogs with possible anti-prostate cancer activity. A series of analogs of anibamine were designed, synthesized and tested against several prostate cancer cell lines. The analogs all acted as CCR5 antagonists at micromolar range affinity to the receptor while their anti-proliferative activity varied depending on the cell line type and their chemical structural properties. Further basal cytotoxicity characterization on these compounds indicated some of them may be suitable for in vivo studies.

Wilms' tumor 1 silencing decreases the viability and chemoresistance of glioblastoma cells in vitro: a potential role for IGF-1R de-repression.

Wilms' tumor 1 (WT1) is a transcription factor with a multitude of downstream targets that have wide-ranging effects in non-glioma cell lines. Though its expression in glioblastomas is now well-documented, the role of WT1 in these tumors remains poorly defined. We hypothesized that WT1 functions as an oncogene to enhance glioblastoma viability and chemoresistance. WT1's role was examined by studying the effect of WT1 silencing and overexpression on DNA damage, apoptosis and cell viability. Results indicated that WT1 silencing adversely affected glioblastoma viability, at times, in synergy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin. To investigate other mechanisms through which WT1 could affect viability, we measured cell cycle distribution, senescence, and autophagy. WT1 silencing had no effect on these processes. Lastly, we examined WT1 regulation of IGF-1R expression. Counterintuitively, upregulation of IGF-1R was evident after WT1 silencing. In conclusion, WT1 functions as a survival factor in glioblastomas, possibly through inhibition of IGF-1R expression.

Amphibian pathogen Batrachochytrium dendrobatidis prevalence is correlated with season and not urbanization in central Virginia.

The global amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been documented among many species throughout the United States, though cases of chytridiomycosis, the resulting disease, have occurred mostly on the west coast. We conducted a 2 yr survey of amphibians along an urban gradient in Virginia, U.S.A., to test whether Bd prevalence among the amphibians sampled varied with urbanization and/or season. A total of 867 adult amphibians from 13 species and 49 tadpoles from 3 species were tested for Bd. The level of urbanization was based on surrounding human population density and anthropogenic disturbance. Bd was detected in 6 species. Bd prevalence was not found to vary with increases in urbanization, but did vary with season. Prevalence peaked in the spring at 45%, when temperatures were between 14 and 25 degrees C, and dropped to below 2% in the autumn. Results from this survey support the hypothesis that Bd is endemic to the studied sites in Virginia. The present study, in concurrence with previous research by other investigators, shows that Bd is affected strongly by weather patterns. Urbanization, defined by human population density, appeared to have minimal impact on the prevalence of Bd. In addition to understanding the geographic distribution of Bd, it is important to understand factors that affect its prevalence if we are to develop approaches to managing this emerging disease.

Anibamine, a natural product CCR5 antagonist, as a novel lead for the development of anti-prostate cancer agents.

Accumulating evidence indicates that the chemokine receptor CCR5 and the chemokine CCL5 may be involved in the proliferation and metastasis of prostate cancer. Consequently, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. As the first natural product CCR5 antagonist, anibamine provides a novel chemical structural skeleton compared with other known antagonists identified through high-throughput screening. Our studies demonstrate that anibamine produces significant inhibition of prostate cancer cell proliferation at micromolar to submicromolar concentrations as well as suppressing adhesion and invasion of the highly metastatic M12 prostate cancer cell line. Preliminary in vivo studies indicate that anibamine also inhibits prostate tumor growth in mice. These findings indicate that anibamine may prove to be a novel lead compound for the development of prostate cancer therapeutic agents.

Identification of a novel cell death receptor mediating IGFBP-3-induced anti-tumor effects in breast and prostate cancer.

Insulin-like growth factor-binding protein-3 (IGFBP-3), a major regulator of endocrine actions of IGFs, is a p53-regulated potent apoptotic factor and is significantly suppressed in a variety of cancers. Recent epidemiologic studies suggest that IGFBP-3 contributes to cancer risk protection in a variety of cancers, and a polymorphic variation of IGFBP-3 influences cancer risk, although other studies vary in their conclusions. Some antiproliferative actions of IGFBP-3 have been reported to be independent of IGFs, but the precise biochemical/molecular mechanisms of IGF-independent, antiproliferative actions of IGFBP-3 are largely unknown. Here we report a new cell death receptor, IGFBP-3R, that is a single-span membrane protein and binds specifically to IGFBP-3 but not other IGFBP species. Expression analysis of IGFBP-3 and IGFBP-3R indicates that the IGFBP-3/IGFBP-3R axis is impaired in breast and prostate cancer. We also provide evidence for anti-tumor effect of IGFBP-3R in vivo using prostate and breast cancer xenografts in athymic nude mice. Further in vitro studies demonstrate that IGFBP-3R mediates IGFBP-3-induced caspase-8-dependent apoptosis in various cancer cells. Knockdown of IGFBP-3R attenuated IGFBP-3-induced caspase activities and apoptosis, whereas overexpression of IGFBP-3R enhanced IGFBP-3 biological effects. IGFBP-3R physically interacts and activates caspase-8, and knockdown of caspase-8 expression or activity inhibited IGFBP-3/IGFBP-3R-induced apoptosis. Here, we propose that IGFBP-3R represents a novel cell death receptor and is essential for the IGFBP-3-induced apoptosis and tumor suppression. Thus, the IGFBP-3/IGFBP-3R axis may provide therapeutic and prognostic value for the treatment of cancer.

Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells.

OBJECT: Wilms tumor 1 (WT1) is overexpressed in many human cancers, including glioblastoma multiforme (GBM). In another study, the authors showed that transient WT1 silencing increases the radiosensitivity of glioma cells. Studies of nonglioma cell lines have demonstrated that WT1 promotes cell proliferation and survival; however, this ability has not been rigorously analyzed in human GBM. METHODS: The authors tested the efficacy of 2 sequences of short hairpin RNA (shRNA) directed against WT1 in U251MG human GBM cells and found that 1 sequence was capable of stably silencing WT1 expression. They then evaluated the effect of WT1 silencing on cellular proliferation, invasion, and in vivo tumor formation. RESULTS: Stable WT1-shRNA expression significantly decreased the proliferation of U251MG cells in vitro as demonstrated by both an adenosine 5'-triphosphate-based viability assay and tritiated thymidine uptake. Furthermore, stable WT1 silencing caused significantly slower growth after the subcutaneous inoculation of tumor cells in the flanks of athymic nude mice and was associated with an increased latency period. CONCLUSIONS: Data in this study provide proof of the principle that downregulation of WT1 causes decreased tumorigenicity of a GBM cell line in vitro and in vivo and suggest that WT1 is a promising target for novel molecular GBM therapies, perhaps in combination with standard treatment modalities.

MicroRNA-17-3p is a prostate tumor suppressor in vitro and in vivo, and is decreased in high grade prostate tumors analyzed by laser capture microdissection.

MicroRNAs (miRs) are a novel class of RNAs with important roles in regulating gene expression. To identify miRs controlling prostate tumor progression, we utilized unique human prostate sublines derived from the parental P69 cell line, which differ in their tumorigenic properties in vivo. Grown embedded in laminin-rich extracellular matrix (lrECM) gels these genetically-related sublines displayed drastically different morphologies correlating with their behaviour in vivo. The non-tumorigenic P69 subline grew as multicellular acini with a defined lumen and basal/polar expression of relevant marker proteins. M12, a highly tumorigenic, metastatic derivative, grew as a disorganized mass of cells with no polarization, whereas the F6 subline, a weakly tumorigenic, non-metastatic M12 variant, reverted to acini formation akin to the P69 cell line. These sublines also differed in expression of vimentin, which was high in M12, but low in F6 and P69 sublines. Analysis of vimentin's conserved 3'-UTR suggested several miRs that could regulate vimentin expression. The lack of miR-17-3p expression correlated with an increase in vimentin synthesis and tumorigenicity. Stable expression of miR-17-3p in the M12 subline reduced vimentin levels 85% and reverted growth to organized, polarized acini in lrECM gels. In vitro motility and invasion assays suggested a decrease in tumorigenic behaviour, confirmed by reduced tumor growth in male athymic, nude mice dependent on miR-17-3p expression. Analysis of LCM-purified clinical human prostatectomy specimens confirmed that miR-17-3p levels were reduced in tumor cells. These results suggest that miR-17-3p functions as a tumor suppressor, representing a novel target to block prostate tumor progression.

Inhibition of vimentin or beta1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo.

Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to beta-catenin, E-cadherin, or alpha6 and beta1 integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via small interfering RNA interference or the expression of alpha6 and beta1integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and beta1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis.

Infection of spotted salamanders (Ambystoma maculatum) with Ichthyophonus-like organisms in Virginia.

Ichthyophonus-like organisms were found in two free-ranging adult spotted salamanders (Ambystoma maculatum) captured within two different vernal ponds in the Virginia Commonwealth University Rice Center for Environmental Life Sciences in Charles City County, Virginia. Histopathologic examination of necropsied specimens revealed large spores, often enclosed by granulomas. These enclosed spores resembled those caused by the fish pathogen Ichthyophonus hoeferi. One salamander displayed an externally visible large swelling beneath the jaws. The other lacked macroscopic abnormalities, but histologic sections of ventral muscle revealed early-stage Ichthyophonus-like organisms and minimal granulomatous reactions. This is the first report of Ichthyophonus-like infection of Ambystoma maculatum in Virginia.

Wilms tumor 1 expression in malignant gliomas and correlation of +KTS isoforms with p53 status.

OBJECT: The WT1 gene is overexpressed in many types of human cancer. It has been demonstrated that Wilms tumor 1 (WT1) promotes tumor cell proliferation and survival in some cell lines by inhibiting p53-mediated apoptosis; however, this relationship has not been investigated in gliomas. The goal in this study was to characterize the expression pattern of WT1 in human gliomas and to determine if a correlation exists between WT1 expression and p53 status. METHODS: The authors screened nine malignant glioma cell lines, 50 glioblastoma multiforme (GBM) samples, and 16 lower-grade glial tumors for WT1 expression. RESULTS: Five of nine cell lines, 44 of 50 GBM samples, and 13 of 16 lower-grade gliomas expressed WT1 mRNA on reverse transcriptase polymerase chain reaction (PCR) analysis. Expression of WT1 was not detected in normal astrocytes. Two WT1 isoforms, +/+ and -/+, were expressed in the majority of these samples. Real-time PCR analysis of the GBM cell lines revealed that the level of WT1 mRNA ranged from 6.33 to 214.70 ng per ng 18S ribosomal RNA. The authors screened the GBM samples for p53 mutation by using PCR and single-stranded conformational polymorphism analysis, and they demonstrated an association between WT1 expression and p53 status. Tumors that contained wild-type p53 were significantly more likely to express WT1 than tumors that contained mutant p53. CONCLUSIONS: The presence of WT1 in glioma cell lines and the majority of primary tumor samples and its absence in normal astrocytes support the suggestion that WT1 expression is important in glioma biology.

Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.

Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.

cDNA microarray analysis identifies genes induced in common by peptide growth factors and androgen in human prostate epithelial cells.

Prostate cancer cells initially require androgen for continued proliferation, but invariably become androgen independent or unresponsive and recur after treatment by androgen ablation. Exploitation of common signaling components downstream of their specific receptors (i.e., androgen receptor (AR), insulin-like growth factor 1 (IGF-1) receptor, and epidermal growth factor (EGF) receptor) could provide a mechanism by which androgen independent cells survive and proliferate. Our objective was to design and implement prostate enriched cDNA microarrays to identify genes induced in prostate epithelial cells in a similar temporal pattern by both androgen and IGF or EGF. AR positive and AR negative human prostate epithelial cells of the M12 line were exposed in parallel to DHT, EGF, or IGF for 0, 6, or 24 h. RNA extracted from each of these groups was analyzed by cDNA microarrays composed of a unique set of 6373 prostate-derived cDNA clones from the Prostate Expression Database (PEDB). We observed statistically significant changes in 20 genes induced in common after 6 and 24 h exposure to androgen or these growth factors, and validated the microarray results by RT-PCR for three or four of these genes: v-myc, isocitrate dehydrogenase, and calnexin. Androgen response element binding motifs were identified in the upstream sequence in 16 of these 20 genes. These results provide comprehensive and unique insights into potential mechanisms by which peptide growth factors provide alternate pathways to control prostate epithelial cell proliferation in malignant states.

Amplification and overexpression of prosaposin in prostate cancer.

We identified prosaposin (PSAP) as a secreted protein expressed in androgen-independent (AI) prostate cancer cells by cloning/sequencing, after probing a PC-3 cDNA library expressed in the lambdaTriplEx phagemid expression vector with a polyclonal rabbit antibody generated against pooled human seminal plasma. PSAP is a neurotrophic molecule; its deficiency or inactivation has proved to be lethal in man and mice, and in mice, it leads to abnormal development and atrophy of the prostate gland, despite normal testosterone levels. We used Southern hybridization, quantitative real-time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis, and we now report the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC-3, DU-145, MDA-PCa 2b, M-12, and NCI-H660. In addition, by using SNP arrays and a set of 25 punch biopsy samples of human prostate cancer xenografts (LAPC3, LuCaP 23.1, 35, 49, 58, 73, 77, 81, 86.2, 92.1, 93, 96, 105, and 115), lymph nodes, and visceral-organ metastases, we detected amplification of the PSAP locus (10q22.1) in LuCaP 58 and 96 xenografts and two lymph node metastases. In addition, AI metastatic prostate cancer cell lines C4-2B and IA8-ARCaP over-expressed PSAP mRNA without evidence of genomic amplification. Taken together with prior data that demonstrated the growth-, migration-, and invasion-promoting activities, the activation of multiple signal transduction pathways, and the antiapoptotic effect of PSAP (or one of its active domains, saposin C) in prostate cancer cells, our current observation of PSAP amplification or overexpression in prostate cancer suggests, for the first time, a role for this molecule in the process of carcinogenesis or cancer progression in the prostate.

Androgen receptor (AR) expression in AR-negative prostate cancer cells results in differential effects of DHT and IGF-I on proliferation and AR activity between localized and metastatic tumors.

BACKGROUND: Two features of the progression from organ-confined to metastatic prostate cancer are dysregulation of the androgen receptor (AR) and a decrease in insulin-like growth factor-type-I receptor (IGF-IR) expression. The purpose of this study was to determine the effect of changes in IGF-IR expression on AR activity. METHODS: M12 human prostate cells were stably transfected with an AR expression construct to produce the M12-AR parental (PAR) cell line. PAR cells were implanted orthotopically into nude mice and M12-AR primary (PRI) cell lines were derived from intraprostatic tumors and metastatic cell lines (MET) were derived from PRI tumors that had metastasized to diaphragm or lung. RESULTS: Tumor formation in the prostate by PAR cells was decreased significantly compared to M12 controls. PAR, PRI, and MET cells expressed equivalent amounts of AR protein; however, IGF-IR expression was increased significantly in PAR and PRI cells. IGF-IR expression decreased in MET lines to the levels seen in M12 control cells. IGF-I significantly enhanced dihydrotestosterone (DHT)-stimulated, but not basal, AR transcriptional activity in PRI cells. In MET cells, IGF-I significantly suppressed DHT-stimulated transcriptional activity. In MET cells in which the IGF-IR was re-expressed from a retroviral vector, the effects of DHT and IGF-I on AR activity were similar to those seen in PRI cells. CONCLUSIONS: This study demonstrates that the changes in IGF-IR expression exhibited by this model of metastatic progression cause significant alterations in AR signaling and suggest that this interaction may be an important aspect of the changes seen in AR function in disease progression in vivo.

Proteomic identification of 14-3-3 sigma as a common component of the androgen receptor and the epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12.

The epidermal growth factor receptor and androgen receptor (AR) both play major roles in the control of prostate growth. Our hypothesis is that shared downstream components of these two signaling pathways are significant participants in androgen-independent growth. Our first objective was to identify proteins whose activation and/or expression in AR-positive prostate epithelial cells are induced by both epidermal growth factor (EGF) and dihydrotestosterone (DHT). AR expression was induced in a tumorigenic, metastatic subline of the SV40 large T-antigen immortalized human prostate epithelial subline M12 by stable transfection with human wild-type AR cDNA. These M12AR (+) cells with functional AR were treated in parallel with EGF (10 ng/ml) or DHT (10(-8) M) for 24 h before 2D gel electrophoresis and Western immunoblotting with antiphosphotyrosine monoclonal antibody. Coomassie blue-stained spots on a 2D gel run in parallel were aligned with the phosphoproteins on the Western immunoblot, and identified by matrix-assisted laser desorption ionization/time-of-flight mass spectroscopy. The most interesting of the seven proteins that appeared to be phosphorylated by these criteria was 14-3-3 protein sigma. Protein extracted after either EGF or DHT treatment, immunoprecipitated with antiphosphotyrosine monoclonal antibody, and immunoblotted by anti-14-3-3 sigma confirmed phosphorylation of 14-3-3 sigma. Addition of either DHT or EGF to the M12AR(+) cells induced subcellular migration of 14-3-3 sigma and activated a 14-3-3 sigma reporter construct. Immunohistochemical analysis revealed nuclear localization of 14-3-3 sigma in higher Gleason grade prostate cancers relative to benign glands. These findings implicate 14-3-3 sigma in the development of human prostate cancer cells and could provide a new target for intervention in prostate cancer.

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.

Proteomic analysis of the tumorigenic human prostate cell line M12 after microcell-mediated transfer of chromosome 19 demonstrates reduction of vimentin.

Critical alterations in proteins that accompany or control the aggressiveness of human prostate cancers remain poorly defined. Previously we demonstrated that the highly tumorigenic, metastatic human prostate cell line M12 was converted to a slow growing, poorly tumorigenic cell line by introduction of an intact human chromosome 19, generating the M12 (F6) hybrid cells. The objective of this report was to identify changes in the protein profile of these M12(F6) microcell hybrid cells. A combination of two-dimensional gel electrophoresis and matrix assisted laser desorption-time of flight-mass spectroscopy was used to compare proteins made by these two cell lines. No consistently increased proteins were identified. However, seven proteins were reproducibly reduced more than twofold: vimentin, hsp90, ATP synthase, 26S protease regulatory subunit, heterogeneous nuclear ribonucleoprotein, T-Complex protein 1 beta, and alpha-1 tubulin. The striking reduction in vimentin protein was accompanied by significantly decreased vimentin mRNA, revealed by Northern blotting. Our findings implicate reduced vimentin in the conversion of these tumorigenic prostate epithelial cells into slow growing, less aggressive cells. These studies demonstrate that application of proteomic analysis to specific problems in an experimental context can yield biologically relevant information about the prostate cancer cell phenotype.

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology.

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.