PC3 prostate tumor-initiating cells with molecular profile FAM65Bhigh/MFI2low/LEF1low increase tumor angiogenesis

<p>Abstract</p> <p>Background</p> <p>Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression.</p> <p>Methods</p> <p>Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3.</p> <p>Results</p> <p>We demonstrate that, under standard culture conditions, ~10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, <it>FAM65B</it>, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, <it>MFI2 </it>and <it>LEF1</it>, that are consistently down regulated. This molecular profile, FAM65B^high/MFI2^low/LEF1^low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin α2β1. PC3 tumors seeded with holoclones showed dramatic down regulation of <it>FAM65B </it>and dramatic up regulation of <it>MFI2 </it>and <it>LEF1</it>, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis.</p> <p>Conclusions</p> <p>These findings support the proposal that PC3 tumors are sustained by a small number of tumor-initiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern <it>FAM65B^high/MFI2^low/LEF1^low</it>. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3.</p

Impact of methoxyacetic acid on mouse Leydig cell gene expression

<p>Abstract</p> <p>Background</p> <p>Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function.</p> <p>Methods</p> <p>Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling.</p> <p>Results</p> <p>A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling.</p> <p>Conclusions</p> <p>These findings on the progressive changes in gene expression induced by MAA in a cultured Leydig cell model may help elucidate signaling pathways that lead to the testicular pathophysiological responses induced by MAA exposure and may identify useful biomarkers of MAA toxicity.</p

Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

<p>Abstract</p> <p>Background</p> <p>Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and <it>via </it>a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery <it>in vivo </it>using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz <it>et al</it>, (2002) Cancer Res. 62: 6928-37).</p> <p>Methods</p> <p>To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase.</p> <p>Results</p> <p>The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 "factory" cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing.</p> <p>Conclusions</p> <p>The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products.</p

Complex modulation of androgen responsive gene expression by methoxyacetic acid

<p>Abstract</p> <p>Background</p> <p>Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional <it>trans</it>-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes <it>in vivo</it>, and which genes might be affected is not known.</p> <p>Methods</p> <p>A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR) was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale.</p> <p>Results</p> <p>MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA with testosterone; these include 16 genes associated with developmental processes, six of which are <it>Hox </it>genes.</p> <p>Conclusions</p> <p>These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model.</p

Posttranscriptional regulation of hepatic NADPH-cytochrome P450 reductase by thyroid hormone: independent effects on poly(A) tail length and mRNA

ABSTRACT Thyroid hormone [triiodothyronine (T3)] positively regulates NADPH cytochrome P450 reductase (P450R) mRNA expression in rat liver, with P450R transcription initiation being a key regulated step. T3 is presently shown to have significant posttranscriptional effects on P450R expression. T3 increased the size of cytoplasmic P450R mRNA by ϳ105 nucleotides 12 h after T3 treatment, followed by a return to basal levels at 24 h. Primer extension analysis and Northern hybridization with 5Ј-untranslated region probes revealed no change in P450R mRNA 5Ј structure with T3 treatment. By contrast, RNase H analysis revealed a transient, T3-induced increase in P450R mRNA poly(A) tail, from ϳ100 to ϳ205 A. This increase in P450R polyadenylation, detectable in the nucleus 8 h after T3 treatment and in the cytoplasm at 12 h, was transient and was reversed by 16 h, when the T3-induced accumulation of cytoplasmic P450R mRNA was near maximal. Actinomycin D blocked the increase in P450R poly(A) tail and the induction of P450R mRNA, indicating a requirement for ongoing gene transcription for both T3 responses. T3 treatment destabilized P450R mRNA in rat liver in vivo, as shown by the T3-dependent 6-fold decrease in cytoplasmic P450R mRNA half-life, from a basal value of Ն16 h in uninduced liver to ϳ2.5 h, measured 24 h after T3 administration. These findings demonstrate that T3 increases nuclear polyadenylation of P450R RNA as a transient, early regulatory response and that this response is temporally dissociated from the subsequent decrease in cytoplasmic P450R mRNA stability

Achieving precise mechanical control in intrinsically noisy systems

How can precise control be realized in intrinsically noisy systems? Here, we develop a general theoretical framework that provides a way of achieving precise control in signal-dependent noisy environments. When the control signal has Poisson or supra-Poisson noise, precise control is not possible. If, however, the control signal has sub-Poisson noise, then precise control is possible. For this case, the precise control solution is not a function, but a rapidly varying random process that must be averaged with respect to a governing probability density functional. Our theoretical approach is applied to the control of straight-trajectory arm movement. Sub-Poisson noise in the control signal is shown to be capable of leading to precise control. Intriguingly, the control signal for this system has a natural counterpart, namely the bursting pulses of neurons-trains of Dirac-delta functions-in biological systems to achieve precise control performance

Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Drug Metab

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily. These transcription factors are predominantly expressed in the liver, where they are activated by structurally diverse compounds, including many drugs and endogenous substances. CAR and PXR regulate the expression of a broad range of genes, which contribute to transcellular transport, bioactivation, and detoxification of numerous xenochemicals and endogenous substances. This article discusses the importance of these receptors for pharmacology and toxicology, emphasizing the role of individual drugs and natural products as agonists, indirect activators, inverse agonists, and antagonists of CAR and PXR

A Search for Correlation of Ultra-High Energy Cosmic Rays with IRAS-PSCz and 2MASS-6dF Galaxies

We study the arrival directions of 69 ultra-high energy cosmic rays (UHECRs) observed at the Pierre Auger Observatory (PAO) with energies exceeding 55 EeV. We investigate whether the UHECRs exhibit the anisotropy signal expected if the primary particles are protons that originate in galaxies in the local universe, or in sources correlated with these galaxies. We cross-correlate the UHECR arrival directions with the positions of IRAS-PSCz and 2MASS-6dF galaxies taking into account particle energy losses during propagation. This is the first time that the 6dF survey is used in a search for the sources of UHECRs and the first time that the PSCz survey is used with the full 69 PAO events. The observed cross-correlation signal is larger for the PAO UHECRs than for 94% (98%) of realisations from an isotropic distribution when cross-correlated with the PSCz (6dF). On the other hand the observed cross-correlation signal is lower than that expected from 85% of realisations, had the UHECRs originated in galaxies in either survey. The observed cross-correlation signal does exceed that expected by 50% of the realisations if the UHECRs are randomly deflected by intervening magnetic fields by 5 degrees or more. We propose a new method of analysing the expected anisotropy signal, by dividing the predicted UHECR source distribution into equal predicted flux radial shells, which can help localise and constrain the properties of UHECR sources. We find that the 69 PAO events are consistent with isotropy in the nearest of three shells we define, whereas there is weak evidence for correlation with the predicted source distribution in the two more distant shells in which the galaxy distribution is less anisotropic.Comment: 23 pages, version published in JCA

Transcriptional induction of hepatic NADPH: cytochrome P450 oxidoreductase by thyroid hormone.

ABSTRACT Studies were carried out to elucidate the mechanism whereby thyroid hormone (T3) induces NADPH:cytochrome P450 oxidoreductase (P450R) mRNA in rat liver in vivo. Northern blot analysis revealed that T3 treatment increases unspliced liver nuclear P450R RNA 4-fold within 8 h and that this induction precedes the induction of mature, cytoplasmic P450R RNA. Unspliced nuclear P450R RNA was suppressed below basal levels 24 h after T3 treatment, despite the continued presence of elevated circulating T3 levels. To determine whether the T3-stimulated increase in nuclear P450R RNA reflects an increase in P450R transcription initiation, nuclear run-on transcription assays were carried out. T3 induced a 6-to 8-fold increase in P450R transcription rate within 12 h, sufficient to account for the observed increase in nuclear P450R precursor RNA, followed by a decrease back to basal transcription levels at 24 h, consistent with the nuclear RNA profile. Similar transcriptional increases were observed in nuclear run-on transcription studies using hybridization probes corresponding to nine different fragments of the P450R gene, spanning exon 2 to exon 16. Thus, P450R transcription initiation, not transcription elongation, is the T3-regulated event. Similar results were obtained during short (5 min) compared with long (45 min) nuclear run-on transcription assays, suggesting that changes in nuclear RNA processing or regulated degradation do not contribute to the overall RNA induction. This finding was confirmed by the ability of the RNA polymerase inhibitor actinomycin D, administered in vivo, to block T3 induction of P450R transcriptional activity. We conclude that P450R transcription, rather than nuclear RNA processing or mRNA stabilization, is the primary mechanism whereby T3 induces hepatic P450R mRNA