Factors related to fish mercury concentrations in Iowa lakes.

Mercury contamination in aquatic ecosystems is a global concern due to the health risks of consuming contaminated fishes. Fish mercury concentrations are influenced by a range of biotic and abiotic factors that vary among regions, but these complex interactions are difficult to disentangle. We collected bluegill (Lepomis macrochirus), white and black crappie (Pomoxis annularis; P. nigromaculatus), largemouth bass (Micropterus salmoides), walleye (Sander vitreus), muskellunge (Esox masquinongy), and northern pike (E. lucius) from waterbodies throughout Iowa and analyzed them for mercury concentrations. We used land use, water chemistry, and fish characteristics to explain variation in mercury concentrations among and within systems. Mercury concentrations were generally low and undetectable (< 0.05 mg/kg) in 43% of fish analyzed. Detected mercury concentrations were highest in largemouth bass, muskellunge, northern pike, and walleye, lowest in black and white crappie and bluegill, and positively related to fish length and age. Mean lake depth, pH, watershed area to lake area ratio, and percent of watershed as open water were positively related to fish mercury concentrations whereas lake area and percent of watershed as agriculture, developed, forested, and grassland were negatively related to mercury concentrations. Additionally, mercury concentrations were higher in shallow natural lakes compared to other lake types. Our results indicate fish mercury concentrations are lower in Iowa lakes compared to other regions. Models we developed in this study can be used to identify other waterbodies that may have elevated mercury concentrations that can guide fish mercury monitoring programs.

Use of occupancy modelling to identify sample sizes and waterbodies with fish exceeding mercury consumption advisory thresholds.

Identifying waterbodies where fish methylmercury concentrations are elevated is critical for development of consumption guidelines. However, mercury concentrations vary among waterbodies and fishes due to a suite of environmental conditions and detection of elevated mercury concentrations is imperfect, resulting in inaccurate consumption guidelines. Occupancy models may be a useful approach for addressing these issues but have not been used for these purposes. Our objectives were to use occupancy modeling to (1) estimate number of samples needed to detect mercury levels surpassing >0.30 mg/kg wet weight in fish at a waterbody (2) identify individual fish-level factors associated with detection probability, and (3) identify environmental-level factors linked to elevated mercury levels in fish at a waterbody. Mercury concentrations were estimated from >500 largemouth bass Micropterus salmoides and walleye Sander vitreus from 30 waterbodies throughout Iowa, USA to identify individuals with concentrations > or <0.30 mg/kg. Probability of detecting mercury concentrations >0.30 mg/kg varied between species and increased with fish length; consequently, more samples were needed to detect elevated mercury concentrations in small versus large fish. The probability of a waterbody having fish with elevated mercury levels increased with the percent grassland and declined with percent agriculture in the watershed, providing prioritization metrics for mercury surveillance programs. Our results demonstrate that occupancy models can be a valuable tool for mercury surveillance due to their ability to estimate necessary sample sizes and identify fish sizes and waterbodies with elevated mercury concentrations while accounting for imperfect detection probabilities.

Factors influencing fish mercury concentrations in Iowa rivers.

Fish mercury concentrations have received considerable attention due to human health implications. Fish mercury concentrations are variable within and among systems due to a suite of biotic and abiotic influences that vary among regions and are difficult to predict. Understanding factors associated with variability in fish mercury concentrations would help guide consumption advisories. Mercury concentrations in channel catfish (Ictalurus punctatus, n = 205), flathead catfish (Pylodictis olivaris, n = 123), northern pike (Esox lucius, n = 60), smallmouth bass (Micropterus dolomieu, n = 176), and walleye (Sander vitreus, n = 176) were assessed in ten Iowa rivers and relationships with land use, water chemistry, and fish characteristics were explored. Mercury concentrations were generally low (mean among all species = 0.17 mg/kg, n = 740) but higher in flathead catfish, northern pike, smallmouth bass, and walleye than channel catfish and were positively related to fish length, age, trophic position, and δ13C signatures. Phosphorus, sulfate, and percent open water and grassland were negatively related to fish mercury concentrations, whereas water hardness, nitrogen-ammonia, Human Threat Index, and percent wetland and forest were positively related to fish mercury concentrations. Fish collected from the Paleozoic Plateau ecoregion in northeast Iowa had higher mercury concentrations than other ecoregions in Iowa. Combined, these factors explained 70% of the variation in fish mercury concentrations. This study provides a comprehensive analysis of abiotic and biotic factors influencing fish mercury concentrations in lotic ecosystems at the individual and system scale that will help guide fish consumption advisories.

A case study and a meta-analysis of seasonal variation in fish mercury concentrations.

Mercury contamination in aquatic ecosystems is a concern due to health risks of consuming fish. Fish mercury concentrations are highly variable and influenced by a range of environmental factors. However, seasonal variation in mercury levels are typically overlooked when monitoring fish mercury concentrations, establishing consumption advisories, or creating accumulation models. Temporal variation in sampling could bias mercury concentration estimates of accumulation potential. Thus, the objectives of this study were to first evaluate seasonal variation of largemouth bass (Micropterus salmoides) axial muscle mercury concentration from two Iowa, USA impoundments. Second, we conducted a meta-analysis to evaluate if seasonal variation in mercury concentration is dependent upon mean mercury concentration, waterbody type, or fish trophic level or mean length. Largemouth bass were collected four times between May and October (24-36 fish per month) from Twelve Mile (2013) and Red Haw (2014) lakes. Largemouth bass axial muscle mercury concentrations were variable within and between lakes, ranging from undetectable ( < 0.05 mg/kg) to 0.54 mg/kg. Largemouth bass mercury concentrations were similar across months in Twelve Mile but varied temporally in Red Haw and were highest in July, intermediate in May and September, and lowest during October. Results of the meta-analysis suggest that seasonal variation in mercury concentrations is more likely to occur as mean mercury concentration of the population increases but is unrelated to waterbody type, trophic status, and fish size. Understanding seasonal variation in fish mercury concentrations will aid in the development of standardized sampling programs for long-term monitoring programs and fish consumption advisories.

Symmetry breaking in spreading RAT2 fibroblasts requires the MAPK/ERK pathway scaffold RACK1 that integrates FAK, p190A-RhoGAP and ERK2 signaling.

The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit.

WhoseEgg: classification software for invasive carp eggs.

The collection of fish eggs is a commonly used technique for monitoring invasive carp. Genetic identification is the most trusted method for identifying fish eggs but is expensive and slow. Recent work suggests random forest models could provide an inexpensive method for identifying invasive carp eggs based on morphometric egg characteristics. While random forests provide accurate predictions, they do not produce a simple formula for obtaining new predictions. Instead, individuals must have knowledge of the R coding language, limiting the individuals who can use the random forests for resource management. We present WhoseEgg: a web-based point-and-click application that allows non-R users to access random forests via a point and click interface to rapidly identify fish eggs with an objective of detecting invasive carp (Bighead, Grass, and Silver Carp) in the Upper Mississippi River basin. This article provides an overview of WhoseEgg, an example application, and future research directions.

PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis.

Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by "switching" interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.

Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Extrinsic interactions in the microenvironment in vivo activate an antiapoptotic multidrug-resistant phenotype in CLL.

The Bcl-2 inhibitor venetoclax has yielded exceptional clinical responses in chronic lymphocytic leukemia (CLL). However, de novo resistance can result in failure to achieve negative minimal residual disease and predicts poor treatment outcomes. Consequently, additional proapoptotic drugs, such as inhibitors of Mcl-1 and Bcl-xL, are in development. By profiling antiapoptotic proteins using flow cytometry, we find that leukemic B cells that recently emigrated from the lymph node (CD69+/CXCR4Low) in vivo are enriched for cell clusters simultaneously overexpressing multiple antiapoptotic proteins (Mcl-1High/Bcl-xLHigh/Bcl-2High) in both treated and treatment-naive CLL patients. These cells exhibited antiapoptotic resistance to multiple BH-domain antagonists, including inhibitors of Bcl-2, Mcl-1, and Bcl-xL, when tested as single agents in a flow cytometry-based functional assay. Antiapoptotic multidrug resistance declines ex vivo, consistent with resistance being generated in vivo by extrinsic microenvironmental interactions. Surviving "persister" cells in patients undergoing venetoclax treatment are enriched for CLL cells displaying the functional and molecular properties of microenvironmentally induced multidrug resistance. Overcoming this resistance required simultaneous inhibition of multiple antiapoptotic proteins, with potential for unwanted toxicities. Using a drug screen performed using patient peripheral blood mononuclear cells cultured in an ex vivo microenvironment model, we identify novel venetoclax drug combinations that induce selective cytotoxicity in multidrug-resistant CLL cells. Thus, we demonstrate that antiapoptotic multidrug-resistant CLL cells exist in patients de novo and show that these cells persist during proapoptotic treatment, such as venetoclax. We validate clinically actionable approaches to selectively deplete this reservoir in patients.

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation.

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Subcellular localization modulates activation function 1 domain phosphorylation in the androgen receptor.

Although the steady-state distribution of the androgen receptor (AR) is predominantly nuclear in androgen-treated cells, androgen-bound AR shuttles between the nucleus and the cytoplasm. In the present study we have addressed how nucleocytoplasmic shuttling contributes to the regulation of AR. Nuclear transport signal fusions were used to force AR localization to the nucleus or cytoplasm of prostate cancer cells, and the effect of localization on shuttling, transcription, androgen binding, and phosphorylation was determined. Fusing the simian virus 40 nuclear localization signal or c-Abl nuclear export signal to AR resulted in androgen-independent localization to the nucleus or cytoplasm, respectively. AR forced to the nucleus was transcriptionally active on prostate-specific antigen and mouse mammary tumor virus promoters driving reporter genes. AR forced to the cytoplasm was largely inactive on the prostate-specific antigen promoter, but, surprisingly, AR was active on the mouse mammary tumor virus promoter and on two endogenous genes examined. Thus, highly transient nuclear localization of AR is sufficient to activate transcription. Androgen dissociation rates and the dissociation constant (K(D)) of AR for androgen were similar whether AR was localized to the cytoplasm or the nucleus, suggesting the ligand-binding cycle of AR is not strictly linked to its compartmentalization. Using phosphosite antibodies, we found that compartmentalization influences the phosphorylation state of AR. We show there is a bias for androgen-dependent phosphorylation of Ser81, Ser256, and Ser308 in the nucleus and androgen-independent phosphorylation of Ser94 in the cytoplasm. We propose that one function of nucleocytoplasmic shuttling is to integrate the signaling environment in the cytoplasm with AR activity in the nucleus.

Receptor for activated C kinase 1 (RACK1) and Src regulate the tyrosine phosphorylation and function of the androgen receptor.

The androgen receptor (AR) remains functionally important in the development and progression of prostate cancer even when the disease seems androgen "independent." Because signal transduction by growth factor receptors increases in advanced prostate cancer and is capable of sensitizing the AR to androgen, there is considerable interest in determining the mechanisms by which signaling systems can modulate AR function. We show herein that the adaptor/scaffolding protein receptor for activated C kinase 1 (RACK1), which was previously reported to interact with the AR, modulates the tyrosine phosphorylation of AR and its interaction with the Src tyrosine kinase. We also show that down-regulation of RACK1 by short interfering RNA inhibits growth and stimulates prostate-specific antigen transcription in androgen-treated prostate cancer cells. Our results suggest that RACK1 mediates the cross-talk of AR with additional binding partners, such as Src, and facilitates the tyrosine phosphorylation and transcriptional activity of the AR.

Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes.

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion.

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.

Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization.

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL.

De novo resistance and rapid recurrence often characterize responses of B-cell malignancies to ibrutinib (IBR), indicating a need to develop drug combinations that block compensatory survival signaling and give deeper, more durable responses. To identify such combinations, we previously performed a combinatorial drug screen and identified the Bcl-2 inhibitor venetoclax (VEN) as a promising partner for combination with IBR in Mantle Cell Lymphoma (MCL). We have opened a multi-institutional clinical trial to test this combination. However, analysis of primary samples from patients with MCL as well as chronic lymphocytic leukemia (CLL) revealed unexpected heterogeneous de novo resistance even to the IBR+VEN combination. In the current study, we demonstrate that resistance to the combination can be generated by microenvironmental agonists: IL-10, CD40L and, most potently, CpG-oligodeoxynucleotides (CpG-ODN), which is a surrogate for unmethylated DNA and a specific agonist for TLR9 signaling. Incubation with these agonists caused robust activation of NF-κB signaling, especially alternative NF-κB, which led to enhanced expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, thus decreasing dependence on Bcl-2. Inhibitors of NF-κB signaling blocked overexpression of these anti-apoptotic proteins and overcame resistance. Inhibitors of Mcl-1, Bcl-xL, or survivin also overcame this resistance, and showed synergistic benefit with the IBR+VEN combination. We conclude that microenvironmental factors, particularly the TLR9 agonist, can generate de novo resistance to the IBR+VEN combination in CLL and MCL cells. This signaling pathway presents targets for overcoming drug resistance induced by extrinsic microenvironmental factors in diverse B-cell malignancies.

Prospective Assessment of Virtual Screening Heuristics Derived Using a Novel Fusion Score.

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

Attenuation of Ras signaling restores androgen sensitivity to hormone-refractory C4-2 prostate cancer cells.

Progression of prostate cancer to androgen-refractory disease is correlated with increased expression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. Many of these growth factor receptors engage Ras as part of their normal signaling activities, raising the possibility that activation of endogenous c-Ras could be a common mechanism for prostate cancer progression. Here we demonstrate that inducible expression of a dominant negative form of Ras restores androgen sensitivity to a hormone-refractory prostate cancer cell line. We show that expression of RasN17 in the hormone-refractory C4-2 cell line enhances in vitro sensitivity to the growth-inhibitory action of the antiandrogen Casodex and inhibits anchorage-independent cell growth. Moreover, although induction of RasN17 by itself has no observable effect on the growth of C4-2 xenografts in intact male mice, it restores androgen dependence to the C4-2 xenografts so that they dramatically regress after surgical androgen ablation.

Constitutive activation of the Ras/mitogen-activated protein kinase signaling pathway promotes androgen hypersensitivity in LNCaP prostate cancer cells.

Progression of prostate cancer ultimately results in a disease that is refractory to hormone ablation therapy but nevertheless continues to require the androgen receptor. Progression to hormone refractory disease is often correlated with overexpression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. Many of these growth factor receptors engage the Ras/mitogen-activated protein (MAP) kinase pathway as part of their signaling activities. This raises the possibility that chronic activation of Ras/MAP kinase signaling could cause or contribute to the progression of prostate cancer. We have demonstrated previously that MAP kinase activation correlates with the progression to advanced hormone refractory disease in patient samples. Here we demonstrate that stable expression of Ras effector-loop mutants that activate the Ras/MAP kinase pathway is sufficient to reduce the androgen requirement of LNCaP prostate cancer cells for growth, prostate-specific antigen expression, and tumorigenicity. We propose that chronic activation of endogenous c-Ras by autocrine and paracrine growth factor stimulation sensitizes the androgen receptor transcriptional complex to subphysiological levels of androgen. This provides a common mechanism for prostate cancer progression driven by diverse agonists.

Smac is required for cytochrome c-induced apoptosis in prostate cancer LNCaP cells.

Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.