Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Unique targeting of androgen-dependent and -independent AR signaling in prostate cancer to overcome androgen resistance.

The androgen receptor (AR) is a major driver of prostate cancer (PCa) and a key therapeutic target for AR inhibitors (ie, Enzalutamide). However, Enzalutamide only inhibits androgen-dependent AR signaling, enabling intrinsic AR activation via androgen-independent pathways, leading to aggressive castration-resistant PCa (CRPC). We investigated the ability of novel anti-cancer agents, Dp44mT and DpC, to overcome androgen resistance. The effect of Dp44mT and DpC on androgen-dependent and independent AR signaling was assessed in androgen-dependent and -independent PCa cells using 2D- and 3D-tissue culture. The clinically trialed DpC was then examined in vivo and compared to Enzalutamide. These agents uniquely promote AR proteasomal degradation and inhibit AR transcription in PCa cells via the upregulation of c-Jun, potently reducing the AR target, prostate-specific antigen (PSA). These agents also inhibited the activation of key molecules in both androgen-dependent and independent AR signaling (ie, EGFR, MAPK, PI3K), which promote CRPC. The clinically trialed DpC also significantly inhibited PCa tumor growth, AR, and PSA expression in vivo, being more potent than Enzalutamide. DpC is a promising candidate for a unique, structurally distinct generation of AR inhibitors that simultaneously target both androgen-dependent and independent arms of AR signaling. No other therapies exhibit such comprehensive and potent AR suppression, which is critical for overcoming the development of androgen resistance.

Cytosolic phospholipase A2α sustains pAKT, pERK and AR levels in PTEN-null/mutated prostate cancer cells.

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A2α (cPLA2α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA2α led to an increase in pAKT, pGSK3ß and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA2α expression with siRNA decreased pAKT, pGSK3ß and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA2α decreased pERK and AR protein levels. The inhibitory effect of cPLA2α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA2α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA2α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.

Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle.

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

Current status of biomarkers for prostate cancer.

Prostate cancer (PCa) is a leading cause of cancer-related death of men globally. Since its introduction, there has been intense debate as to the effectiveness of the prostate specific antigen (PSA) test as a screening tool for PCa. It is now evident that the PSA test produces unacceptably high rates of false positive results and is not prognostic. Here we review the current status of molecular biomarkers that promise to be prognostic and that might inform individual patient management. It highlights current efforts to identify biomarkers obtained by minimally invasive methods and discusses current knowledge with regard to gene fusions, mRNA and microRNAs, immunology, and cancer-associated microparticles.

The Importance of Experimental Investigation of the Peripheral Oxytocin System.

Oxytocin and oxytocin receptors are synthesized in the periphery where paracrine/autocrine actions have been described alongside endocrine actions effected by central release of oxytocin from the posterior pituitary. In the female reproductive system, classical actions of uterine contraction and milk ejection from mammary glands are accompanied by actions in the ovaries where roles in steroidogenesis, follicle recruitment and ovulation have been described. Steroidogenesis, contractile activity, and gamete health are similarly affected by oxytocin in the male reproductive tract. In the cardiovascular system, a local oxytocinergic system appears to play an important cardio-protective role. This role is likely associated with emerging evidence that peripheral oxytocin is an important hormone in the endocrinology of glucose homeostasis due to its actions in adipose, the pancreas, and the largely ignored oxytocinergic systems of the adrenal glands and liver. Gene polymorphisms are shown to be associated with a number of reported traits, not least factors associated with metabolic syndrome.

Oxytocin stimulates lipolysis, prostaglandin E2 synthesis, and leptin secretion in 3T3-L1 adipocytes.

A model of oxytocin in the regulation of metabolic status has described one of oxytocin synthesis and release from the neurohypophysis in response to leptin, to suppress further leptin release. In addition, a lipogenic role for oxytocin has been suggested, consistent with an insulinergic action. This model, however, may be incorrect. Oxytocin reduces fat mass in the absence of either leptin or leptin receptor signalling, thereby challenging the interdependence between leptin and oxytocin. An oxytocin induced production of the anti-lipolytic prostaglandin E2 (PGE2) might account for this. Media from 3T3-L1 differentiated adipocytes treated with oxytocin (0-50 nmol.L-1) for 24 hrs were assayed for PGE2, leptin, adiponectin, and glycerol. Harvested cells were analysed for lipid droplet triglyceride and cytosolic free fatty acid (FFA) by flow cytometry, and for altered expression of lipolytic and lipogenic associated gene ontology transcripts by cDNA array. Both PGE2 and leptin secretion were significantly increased by oxytocin treatment whilst adiponectin secretion was not. A significant increase in cytosolic FFA was detected following oxytocin treatment, similar to that determined following treatment with isoproterenol (positive control). A significant increase in glycerol release to the culture media confirmed a lipolytic effect. No enrichment of lipolytic and lipogenic associated gene ontology transcripts was determined, but significant overrepresentation of chemosensory olfactory transcripts was. In conclusion, oxytocin stimulates lipolysis in 3T3-L1 adipocytes, mediated by autocrine/paracrine actions of PGE2 and leptin. To confirm that this response is mediated solely by the oxytocin receptor, further experiments would require those effects being blocked by a specific oxytocin antagonist.

A novel splice variant of the beta-tropomyosin (TPM2) gene in prostate cancer.

Decreased expression of high molecular weight isoforms of tropomyosin (Tm) is associated with oncogenic transformation and is evident in cancers, with isoform Tm1 seemingly an important tumor suppressor. Tm1 expression in prostate cancer has not previously been described. In this study, while demonstrating suppressed levels of Tm1 in the prostate cancer cell lines LNCaP, PC3, and DU-145 compared to normal prostate epithelial cell primary isolates (PrEC), a novel splice variant of the TPM2 gene was identified. Quantitative RT-PCR determined significantly greater levels of the transcript variant in all three prostate cancer cell lines than in normal prostate epithelial cells. Characterization of this novel variant demonstrated it to include exon 6b, previously thought unique to the muscle-specific beta-Tm isoform, with an exon arrangement of 1-2-3-4-5-6a-6b-7-8-10. Inclusion of exon 6b introduces a premature stop codon directly following the 6a-6b exon boundary. Western blot analysis demonstrated the presence of a truncated protein in prostate cancer cell lines that was absent in normal prostate epithelial cells. It is hypothesized that this truncated protein will result in suppression of Tm1 polymer formation required for actin filament association. The lack of Tm polymer-actin association will result in loss of the stable actin microfilament organization and stress fiber formation, a state associated with cell transformation.

Expression of the actin-associated protein transgelin (SM22) is decreased in prostate cancer.

Transgelin is an actin-binding protein shown to be tumour-suppressive. Loss of transgelin expression in transformed cells is associated with oncogenesis. This study aimed to determine whether transgelin expression was suppressed in prostate cancer. An in silico meta-analysis with public-domain expressed-sequence-tag libraries of normal human prostate epithelium, prostatic intraepithelial neoplasia, invasive carcinoma and metastasised lesions predicted decreased transgelin expression with disease progression. Similarly, analysis of Affymetrix gene chip data and the Oncomine database indicated that transgelin was one the 2% most significant of all down-regulated genes in response to prostate cancer. Analysis by quantitative reverse transcription with the polymerase chain reaction (qRT-PCR) of patient biopsies determined transgelin expression to be significantly lower in prostate tumour tissue than in matched normal tissue. Similarly, qRT-PCR and Western blot analysis of representative prostate cancer cell lines demonstrated significantly lower levels of transgelin mRNA and protein in all but the DU145 prostate cancer cell line. Increased expression of TAGLN and increased transgelin protein in response to treatment with transforming growth factor-beta suggested that reduced expression in prostate cancer was not attributable to gene promoter suppression by hypermethylation. Gene ontology function analysis highlighted the importance of transgelin in the co-deregulation of actin-binding proteins. Thus, transgelin is suppressed during prostate cancer progression and seems to be an important factor in the dysregulation of the actin cytoskeleton.

The TGF-beta, PI3K/Akt and PTEN pathways: established and proposed biochemical integration in prostate cancer.

A key to the development of improved pharmacological treatment strategies for cancer is an understanding of the integration of biochemical pathways involved in both tumorigenesis and cancer suppression. Furthermore, genetic markers that may predict the outcome of targeted pharmacological intervention in an individual are central to patient-focused treatment regimens rather than the traditional 'one size fits all' approach. Prostate cancer is a highly heterogeneous disease in which a patient-tailored care program is a holy grail. This review will describe the evidence that demonstrates the integration of three established pathways: the tumour-suppressive TGF-beta (transforming growth factor-beta) pathway, the tumorigenic PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) pathway and the tumour-suppressive PTEN (phosphatase and tensin homologue deleted on chromosome 10) pathway. It will discuss gene polymorphisms and somatic mutations in relevant genes and highlight novel pharmaceutical agents that target key points in these integrated pathways.

Pharmacological targeting of the integrated protein kinase B, phosphatase and tensin homolog deleted on chromosome 10, and transforming growth factor-beta pathways in prostate cancer.

Prostate cancer is a highly heterogenous disease in which a patient-tailored care program is much desired. Central to this goal is the development of novel targeted pharmacological interventions. To develop these treatment strategies, an understanding of the integration of cellular pathways involved in both tumorigenesis and tumor suppression is crucial. Of further interest are the events elicited by drug treatments that exploit the underlying molecular pathology in cancer. This review briefly describes the evidence that suggests integration of three established pathways: the tumorigenic phosphoinositide 3-kinase/protein kinase B (AKT) pathway, the tumor suppressive phosphatase and tensin homolog deleted on chromosome 10 pathway, and the tumor suppressive transforming growth factor-beta pathway. More importantly, we discuss novel pharmaceutical agents that target key points of integration in these three pathways. These new therapeutic strategies include the use of agents that target iron to inhibit proliferation via multiple mechanisms and suppression of AKT by cytosolic phospholipase A(2)-alpha inhibitors.

Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.

The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution of biologically pertinent questions. Here a method for differentiating urothelium from mouse embryonic stem cells in chemically defined conditions is described. The method includes a description of flow cytometry and RT-PCR analysis of definitive endoderm markers Cxcr4, c-Kit, and FoxA2, and of terminally differentiated urothelial cell markers Upk1b and Upk2.

A Promising Future for Prostate Cancer Diagnostics.

It has been estimated that globally there is a death attributable to prostate cancer every four minutes. As life expectancy in all world regions increases, so too incidence of this disease of the ageing male will increase. For many men diagnosis occurs after presentation with symptoms of altered urinary dynamics. Unfortunately, these changes, whilst also associated with benign disease, are evident quite late in the aetiology of prostate cancer. Early detection provides for better management and prognosis. This Special Issue provides an up to date view of the advances made towards early diagnosis and prognosis. It provides reviews of advanced imaging techniques (e.g., multiparametric MRI and protocols), and of biomaterials and molecular biomarkers currently being explored (e.g., microRNAs, proteomics) and the technologies that are revolutionizing this field. It describes the multi-disciplinary approaches that are essential to inexpensive, deliverable and accurate platforms for prostate cancer diagnostics.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots.

Understanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level.

Cosuppression of Sprouty and Sprouty-related negative regulators of FGF signalling in prostate cancer: a working hypothesis.

Deregulation of FGF receptor tyrosine kinase (RTK) signalling is common in prostate cancer. Normally, to moderate RTK signalling, induction of Sprouty (SPRY) and Sprouty-related (SPRED) antagonists occurs. Whilst decreased SPRY and SPRED has been described in some cancers, their role in prostate cancer is poorly understood. Therefore, we hypothesise that due to the need for tight regulation of RTK signalling, SPRY and SPRED negative regulators provide a degree of redundancy which ensures that a suppression of one or more family member does not lead to disease. Contrary to this, our analyses of prostates from 24-week-old Spry1- or Spry2-deficientmice, either hemizygous (+/-) or homozygous (-/-) for the null allele, revealed a significantly greater incidence of PIN compared to wild-type littermates. We further investigated redundancy of negative regulators in the clinical setting in a preliminary analysis of Gene Expression Omnibus and Oncomine human prostate cancer datasets. Consistent with our hypothesis, in two datasets analysed a significant cosuppression of SPRYs and SPREDs is evident. These findings demonstrate the importance of negative regulators of receptor tyrosine signalling, such as Spry, in the clinical setting, and highlight their importance for future pharmacopeia.

Oxytocin differentially effects 3ß-hydroxysteroid dehydrogenase and 5α-reductase activities in prostate cancer cell lines.

It is known that oxytocin stimulates steroidogenesis in several organs by modulating activity of 3ß-hydroxysteroid dehydrogenases (HSD3B) and steroid 5α-reductases (SRD5A). However, this has not been established in prostate cancer where these enzymes, key to local production of androgens, are increased. Analysis of both HSD3B and SRD5A activities using a live cell in situ colourimetric assay demonstrated that in PC-3 cells HSD3B activity was significantly increased by oxytocin whilst SRD5A activity was unchanged. This was confirmed in ELISA based assays of conversion of pregnenolone to progesterone and testosterone to dihydrotestosterone in cell lysates following treatment. In contrast, oxytocin significantly inhibited HSD3B activity in LNCaPs, but significantly increased activity of SRD5A, as confirmed by ELISA assays. Analysis of both cell lines by microarray and qRT-PCR determined that these changes were not due to altered gene transcription. This study demonstrates differential effects of oxytocin on the activities of key de novo steroidogenic enzymes in prostate cancer cells.

The mammalian copper transporters CTR1 and CTR2 and their roles in development and disease.

Copper is vital to cell function. The influx of reduced copper ions is controlled by two functionally homologous transmembrane solute carrier transporters CTR1 (encoded by SLC31A1) and CTR2 (encoded by SLC31A2). These copper transporters vary in their expression profiles and intracellular localisation patterns. CTR1 plays roles in the developing embryo as well as regulating homeostasis in the adult mammal. In contrast, the regulation, expression and function of CTR2 is poorly defined. Both are capable of transporting other divalent metal ions and are the primary transporters for platinum-based chemotherapeutic drugs such as cisplatin. This review summarises our current understanding of these two copper transporters and highlights their roles in cellular processes, embryonic development, differentiation, cancer, immunity and disease.

Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells.

NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT-PCR (reverse transcriptase-PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1-49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49-Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.

Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach.

BACKGROUND: The haemotropic mycoplasmas Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum cause feline infectious anaemia with infection rates in feline populations reflecting widespread subclinical infection. Clinically significant infections are much rarer but can be life-threatening. Current diagnosis is dependent upon visualising organisms in stained blood smears, PCR or quantitative PCR (qPCR). These procedures are labour-intensive and time-consuming. Furthermore, PCR-based approaches offer limited insight into the disease burden of the infected animal. METHODS: We have developed a novel and rapid flow cytometric system that permits diagnosis of haemotropic mycoplasma infections and quantitation of the percentage of erythrocytes that are parasitized. The method exploits the fact that mature mammalian erythrocytes, the host cell for haemoplasmas, are enucleated and thus lack nucleic acid. DRAQ5 is a synthetic anthrocycline dye which rapidly crosses cell membranes and binds to nucleic acids. The presence of exogenous bacterial DNA in mammalian erythrocytes can, therefore, be detected by DRAQ5 uptake and flow cytometric detection of DRAQ5 fluorescence. RESULTS: Here, we show that this system can detect epi-erythrocytic infection of companion felines by haemotropic mycoplasma. Due to their differences in size, and hence the quantity of DNA, the two major feline hemoplasmas M. haemofelis and Candidatus M. haemominutum can be distinguished according to DRAQ5 fluorescence. We have also shown the usefulness of DRAQ5 uptake in monitoring a cat infected with M. haemofelis sequentially during treatment with doxycycline. CONCLUSIONS: The technique described is the first report of a flow cytometric method for detecting haemotropic mycoplasmas in any species and could be applied to widespread screening of animal populations to assess infection by these epi-erythrocytic parasites.

Does TGF-ß induced formation of actin stress fibres reinforce Smad dependent TGF-ß signalling in the prostate?

In the normal prostate, and during early stages of prostate cancer (PCa) development, the cytokine transforming growth factor beta (TGF-ß) acts as a tumour suppressor by inducing cytostasis and apoptosis. However, during tumour development these Smad signalling-dependent endpoints are lost in favour of Smad-independent tumourigenic actions of TGF-ß. In this working hypothesis we present an argument for an intimate association between the TGF-ß signalling pathway and the actin cytoskeleton that acts to reinforce the tumour suppressive actions of TGF-ß in the normal prostate epithelial cell. The rationale is that TGF-ß induces expression of the actin binding and stabilising proteins transgelin and tropomyosin. Expression of these proteins is progressively repressed during PCa development, and is inhibited by constitutive activation of the Ras/MEK/ERK pathway, also known to antagonise TGF-ß tumour suppression in PCa. The subsequent de-stabilisation of the actin cytoskeleton might, therefore, result in suppression of TGF-ß/Smad signalling as an intact link between cytoskeleton and TGF-ß receptor/Smad complex is essential. Filamin A is a scaffold protein that provides this link for receptor associated Smads. It is required for activation of the TGF-ß signal transduction pathway. Thus, actin filament disorganisation would prevent Filamin A/R-Smad mediated TGF-ß signalling, a subsequent loss of tumour suppression and hence promote the progression of PCa. Furthermore, it could be one mechanism by which the switch to a TGF-ß tumourigenic response occurs.