Glycoamino Acid Analogues of the Thomsen-Friedenreich Tumor-Associated Carbohydrate Antigen: Synthesis and Evaluation of Novel Antiproliferative Factor Glycopeptides.

Glycoamino acid analogues of the Thomsen-Friedenreich antigen disaccharide, where the 4' and 4″ hydroxyl groups were substituted with fluorine or hydrogen, were synthesized and incorporated into the asialylated antiproliferative factor (as-APF), a biologically active form of APF, a glycopeptide found in the urine of patients with interstitial cystitis. Various strategies were employed to incorporate the fluorine atom at the 4-positions of either the galactose or N-acetylgalactosamine unit of the disaccharide antigen, based on stereochemistry and reactivity. These glycopeptides were evaluated in antiproliferative assays on both primary normal bladder epithelial cells and T24 bladder carcinoma cells. Unlike many previously published substitutions to APF, mono-4'-fluorination of the GalNAc residue did not affect the activity, whereas fluoro-derivatives of the galactose 4″-position or both 4' and 4″ hydroxyls showed a reduced potency relative to the monosubstituted GalNAc derivative. A fourth compound where the 4″ position of galactose was deoxygenated showed a lower potency than the parent and monosubstituted compounds. These results suggest that specific substitutions in the sugar moieties in the APF can be tolerated, and the glycomimetic design of APF analogues can include fluorine in the GalNAc sugar of the disaccharide.

Death-associated Protein Kinase-1 Expression and Autophagy in Chronic Lymphocytic Leukemia Are Dependent on Activating Transcription Factor-6 and CCAAT/Enhancer-binding Protein-ß.

Expression of DAPK1, a critical regulator of autophagy and apoptosis, is lost in a wide variety of tumors, although the mechanisms are unclear. A transcription factor complex consisting of ATF6 (an endoplasmic reticulum-resident factor) and C/EBP-ß is required for the IFN-γ-induced expression of DAPK1 IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-ß are obligatory for the formation of this transcriptional complex. We report that defects in this pathway fail to control growth of chronic lymphocytic leukemia (CLL). Consistent with these observations, IFN-γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 and/or C/EBP-ß. Together, these results identify a molecular basis for the loss of DAPK1 expression in CLL.

Abnormal Akt signalling in bladder epithelial cell explants from patients with interstitial cystitis/bladder pain syndrome can be induced by antiproliferative factor treatment of normal bladder cells.

OBJECTIVES: To determine whether protein kinase B (Akt) signalling and secretion of specific downstream effector proteins are abnormal in specific cell fractions of bladder epithelial cells from patients with interstitial cystitis/bladder pain syndrome (IC/BPS), as explanted bladder epithelial cells from patients with IC/BPS produce a frizzled 8-related glycopeptide antiproliferative factor (APF) that inhibits normal bladder epithelial cell proliferation and expression of several proteins known to be regulated by Akt signalling. A related secondary objective was to determine whether treatment of normal bladder epithelial cells with active synthetic asialo-antiproliferative factor (as-APF) induces similar changes in Akt signalling and specific downstream effector proteins/mRNAs. PATIENTS AND METHODS: Cell proteins were extracted into four subcellular fractions from primary bladder epithelial explants of six patients who fulfilled modified National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria for IC/BPS and six age- and gender-matched controls. Total and/or phosphorylated cellular Akt, glycogen synthase kinase 3ß (GSK3ß), and ß-catenin; total cellular JunB; and secreted matrix metalloproteinase 2 (MMP2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) levels were determined by Western blot. MMP2, JunB, p53, uroplakin 3 (UPK3), and ß-actin mRNAs were quantified by quantitative reverse transcriptase-polymerase chain reaction. Akt activity was determined by nonradioactive assay. RESULTS: IC/BPS cells had lower Akt activity, along with lower Akt ser473- and GSK3ß ser9-phosphorylation and higher ß-catenin ser33,37/thr41-phosphorylation in specific fractions as compared with matched control cells. IC/BPS explants also had evidence of additional downstream abnormalities compared with control cells, including lower nuclear JunB; lower secreted MMP2 and HB-EGF; plus lower MMP2, JunB, and UPK3 mRNAs but higher p53 mRNA relative to ß-actin. Each of these IC/BPS cell abnormalities was also induced in normal cells by as-APF. CONCLUSION: These findings indicate that IC/BPS cells have abnormal Akt activity with downstream protein expression abnormalities including decreased MMP2 and HB-EGF secretion. They also support the hypothesis that APF plays a role in the pathogenesis of IC/BPS via its effects on cell Akt signalling and HB-EGF production.

Varicella-Zoster Virus Vasculopathy: A Case Report Demonstrating Vasculitis using Black-Blood MRI.

Infections are rare but important causes of stroke. Among these, varicella zoster virus has been known to cause ischemic stroke. During an attack of herpes zoster ophthalmicus, it has been hypothesized that the virus replicates in the trigeminal ganglion and travels via the trigeminal nerve centrally to cause cerebral vasculopathy. Here we present a case of a 69 year-old Caucasian immunocompromised woman who suffered recurrent ischemic infarcts within the same vascular distribution following an episode of zoster ophthalmicus three months prior. An imaging technique termed black-blood magnetic resonance imaging was utilized to aid in the diagnosis of cerebral vasculitis. The case is used to provide a literature review of the pathogenesis, diagnosis, and treatment of cerebral varicella zoster vasculopathy. In situations where an isolated unilateral cerebral vasculopathy is identified, neurologists are urged to consider varicella zoster as a treatable etiologic agent, as untreated vasculopathy can lead to further strokes.

Evidence for bladder urothelial pathophysiology in functional bladder disorders.

Understanding of the role of urothelium in regulating bladder function is continuing to evolve. While the urothelium is thought to function primarily as a barrier for preventing injurious substances and microorganisms from gaining access to bladder stroma and upper urinary tract, studies indicate it may also function in cell signaling events relating to voiding function. This review highlights urothelial abnormalities in bladder pain syndrome/interstitial cystitis (BPS/IC), feline interstitial cystitis (FIC), and nonneurogenic idiopathic overactive bladder (OAB). These bladder conditions are typified by lower urinary tract symptoms including urinary frequency, urgency, urgency incontinence, nocturia, and bladder discomfort or pain. Urothelial tissues and cells from affected clinical subjects and asymptomatic controls have been compared for expression of proteins and mRNA. Animal models have also been used to probe urothelial responses to injuries of the urothelium, urethra, or central nervous system, and transgenic techniques are being used to test specific urothelial abnormalities on bladder function. BPS/IC, FIC, and OAB appear to share some common pathophysiology including increased purinergic, TRPV1, and muscarinic signaling, increased urothelial permeability, and aberrant urothelial differentiation. One challenge is to determine which of several abnormally regulated signaling pathways is most important for mediating bladder dysfunction in these syndromes, with a goal of treating these conditions by targeting specific pathophysiology.

Monoallelic loss of tumor suppressor GRIM-19 promotes tumorigenesis in mice.

Gene-associated with retinoid-interferon induced mortality-19 (GRIM-19), a STAT3-inhibitory protein, was isolated as a growth-suppressive gene product using a genome-wide expression knockdown screen. We and others have shown a loss of expression and occurrence of mutations in the GRIM-19 gene in a variety of primary human cancers, indicating its potential role as tumor suppressor. To help investigate its role in tumor development in vivo, we generated a genetically modified mouse in which Grim-19 can be conditionally inactivated. Deletion of Grim-19 in the skin significantly increased the susceptibility of mice to chemical carcinogenesis, resulting in development of squamous cell carcinomas. These tumors had high Stat3 activity and an increased expression of Stat3-responsive genes. Loss of Grim-19 also caused mitochondrial electron transport dysfunction resulting from failure to assemble electron transport chain complexes and altered the expression of several cellular genes involved in glycolysis. Surprisingly, the deletion of a single copy of the Grim-19 gene was sufficient to promote carcinogenesis and formation of invasive squamous cell carcinomas. These observations highlight the critical role of GRIM-19 as a tumor suppressor.

Safety of zoster vaccine in elderly adults following documented herpes zoster.

BACKGROUND: After completion of the Shingles Prevention Study (SPS; Department of Veterans Affairs Cooperative Studies Program Number 403), SPS participants who had initially received placebo were offered investigational zoster vaccine without charge. This provided an opportunity to determine the relative safety of zoster vaccine in older adults following documented herpes zoster (HZ). METHODS: A total of 13 681 SPS placebo recipients who elected to receive zoster vaccine were followed for serious adverse events (SAE) for 28 days after vaccination. In contrast to the SPS, a prior episode of HZ was not a contraindication to receiving zoster vaccine. The SPS placebo recipients who received zoster vaccine included 420 who had developed documented HZ during the SPS. RESULTS: The mean interval between the onset of HZ and the receipt of zoster vaccine in the 420 recipients with prior HZ was 3.61 years (median interval, 3.77 years [range, 3-85 months]); the interval was <5 years for approximately 80% of recipients. The proportion of vaccinated SPS placebo recipients with prior HZ who developed ≥ 1 SAE (0.95%) was not significantly different from that of vaccinated SPS placebo recipients with no prior history of HZ (0.66%), and the distribution of SAEs in the 2 groups was comparable. CONCLUSIONS: These results demonstrate that the general safety of zoster vaccine in older persons is not altered by a recent history of documented HZ, supporting the safety aspect of the Centers for Disease Control and Prevention Advisory Committee on Immunization Practices recommendation to administer zoster vaccine to all persons ≥ 60 years of age with no contraindications, regardless of a prior history of HZ.

A synthetic form of frizzled 8-associated antiproliferative factor enhances p53 stability through USP2a and MDM2.

Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.

Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

UNLABELLED: What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. OBJECTIVE: • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. MATERIALS AND METHODS: • To identify more differentially expressed genes with a lower false discovery rate from a previously published microarray data set, an integrative hypothesis-testing statistical approach was applied. • For validation experiments, expression and phosphorylation levels of select proteins were evaluated by western blotting. RESULTS: • Integration analysis of this transcriptomics data set with our own quantitative proteomics data set identified 10 genes that are potentially regulated by APF in vivo from 4140 differentially expressed genes identified with a false discovery rate of 1%. • Of these, five (i.e. JUP, MAPKSP1, GSPT1, PTGS2/COX-2 and XPOT) were found to be prominent after network modelling of the common genes identified in the proteomics and microarray studies. • This molecular signature reflects the biological processes of cell adhesion, cell proliferation and inflammation, which is consistent with the known physiological effects of APF. • Lastly, we found the mammalian target of rapamycin pathway was down-regulated in response to APF. CONCLUSION: • This unbiased integration analysis of in vitro quantitative proteomics data with in vivo quantitative transcriptomics data led to the identification of potential downstream mediators of the APF signal transduction pathway.

Quantitative proteomics identifies a beta-catenin network as an element of the signaling response to Frizzled-8 protein-related antiproliferative factor.

Antiproliferative factor (APF), a Frizzled-8 protein-related sialoglycopeptide involved in the pathogenesis of interstitial cystitis, potently inhibits proliferation of normal urothelial cells as well as certain cancer cells. To elucidate the molecular mechanisms of the growth-inhibitory effect of APF, we performed stable isotope labeling by amino acids in cell culture analysis of T24 bladder cancer cells treated with and without APF. Among over 2000 proteins identified, 54 were significantly up-regulated and 48 were down-regulated by APF treatment. Bioinformatic analysis revealed that a protein network involved in cell adhesion was substantially altered by APF and that ß-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated ß-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of ß-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable ß-catenin rescued growth inhibition in response to APF, confirming that ß-catenin is a key mediator of APF signaling. Notably, the key role of ß-catenin in APF signaling is not restricted to T24 cells, but was also observed in an hTERT-immortalized human bladder epithelial cell line, TRT-HU1. In addition, the network model suggested that ß-catenin is linked to cyclooxygenase-2 (COX-2), implying a potential connection between APF and inflammation. Functional assays verified that APF increased the production of prostaglandin E(2) and that down-modulation of ß-catenin elevated COX-2 expression, whereas forced expression of nondegradable ß-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that ß-catenin was down-regulated whereas COX-2 was up-regulated in epithelial cells explanted from IC bladder biopsies compared with control tissues. In summary, our quantitative proteomics study describes the first provisional APF-regulated protein network, within which ß-catenin is a key node, and provides new insight that targeting the ß-catenin signaling pathway may be a rational approach toward treating interstitial cystitis.

An hTERT-immortalized human urothelial cell line that responds to anti-proliferative factor.

Studies of the urothelium, the specialized epithelial lining of the urinary bladder, are critical for understanding diseases affecting the lower urinary tract, including interstitial cystitis, urinary tract infections and cancer. However, our understanding of urothelial pathophysiology has been hampered by a lack of appropriate model systems. Here, we describe the isolation and characterization of a non-transformed urothelial cell line (TRT-HU1), originally explanted from normal tissue and immortalized with hTERT, the catalytic subunit of telomerase. We demonstrate responsiveness of the cells to anti-proliferative factor (APF), a glycopeptide implicated in the pathogenesis of interstitial cystitis. TRT-HU1 carries a deletion on the short arm of chromosome 9, an early genetic lesion in development of bladder cancer. TRT-HU1 urothelial cells displayed growth and migration characteristics similar to the low-grade papilloma cell line RT4. In contrast, we observed marked differences in both phenotype and gene expression profiles between TRT-HU1 and the highly malignant T24 cell line. Together, these findings provide the first demonstration of a non-transformed, continuous urothelial cell line that responds to APF. This cell line will be valuable for studies of both benign and malignant urothelial cell biology.

Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells.

BACKGROUND: Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. METHODS: T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3ß (GSK3ß), ß-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3ß, MMP2, ß-catenin, and p53 protein expression, plus Akt, GSK-3ß, and ß-catenin phosphorylation, were determined by Western blot. RESULTS: T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3ß tyr216 phosphorylation, and ß-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and ß-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3ß, or ß-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3ß/ß-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown. CONCLUSIONS: Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3ß, and ß-catenin) plus mRNA and protein expression of p53 and MMP2.

Structure-Activity Studies on Antiproliferative Factor (APF) Glycooctapeptide Derivatives.

Antiproliferative factor (APF), a sialylated glycopeptide secreted by explanted bladder epithelial cells from interstitial cystitis/painful bladder syndrome (IC/PBS) patients, and its unsialylated analogue (as-APF) significantly decrease proliferation of bladder epithelial cells and/or certain carcinoma cell lines in vitro. We recently reported a structure-activity relationship profile for the peptide portion of as-APF and revealed that truncation of the C-terminal alanine did not significantly affect antiproliferative activity. To better understand the structural basis for the maintenance of activity of this truncated eight amino acid as-APF (as-APF8), we synthesized several amino acid-substituted derivatives and studied their ability to inhibit bladder epithelial cell proliferation in vitro as well as their solution conformations by CD and NMR spectroscopy. While single amino acid changes to as-APF8 often strongly reduced activity, full potency was retained when the trivaline tail was replaced with three alanines. The Ala(6-8) derivative 9 is the simplest, fully potent APF analogue synthesized to date.

Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling.

Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.

Heparin-binding epidermal growth factor-like growth factor functionally antagonizes interstitial cystitis antiproliferative factor via mitogen-activated protein kinase pathway activation.

OBJECTIVE: To delineate the mechanism underlying the potential functional relationship between interstitial cystitis antiproliferative factor (APF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF), as APF has previously been shown to decrease the proliferation rate of normal bladder epithelial cells and the amount of HB-EGF produced by these cells. MATERIALS AND METHODS: APF-responsive T24 transitional carcinoma bladder cells were treated with high-pressure liquid chromatography-purified native APF with or without HB-EGF to determine the involvement of signalling pathways and proliferation by Western blot analysis, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (Erk)/MAPK assays, and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Cyclic stretch induced the secretion of HB-EGF from T24 cells overexpressing the HB-EGF precursor, resulting in enhanced proliferation. T24 cells treated with APF had increased p38MAPK activity and suppressed cell growth, events that were both reversed by treatment with a p38MAPK-selective inhibitor. Activation of Erk/MAPK by HB-EGF was inhibited by APF, and APF did not stimulate p38MAPK in the presence of soluble HB-EGF or when cells overexpressed constitutively secreted HB-EGF. Lastly, APF inhibitory effects on cell growth were attenuated by HB-EGF. CONCLUSIONS: These results indicate that HB-EGF and APF are functionally antagonistic and signal through parallel MAPK signalling pathways in bladder cells.

Structure-activity relationship studies for the peptide portion of the bladder epithelial cell antiproliferative factor from interstitial cystitis patients.

We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of (3)H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.

Urine markers do not predict biopsy findings or presence of bladder ulcers in interstitial cystitis/painful bladder syndrome.

PURPOSE: We tested for associations between urine markers, bladder biopsy features and bladder ulcers in interstitial cystitis/painful bladder syndrome. MATERIALS AND METHODS: Subjects were 72 patients with interstitial cystitis/painful bladder syndrome undergoing bladder distention and biopsy. Urine was collected before the procedure. Urine marker levels were correlated with biopsy and cystoscopic findings. Patients with no previous interstitial cystitis/painful bladder syndrome treatments (47) were analyzed separately from previously treated patients (25). RESULTS: For untreated patients urine interleukin-6 and cyclic guanosine monophosphate were associated with urothelial epidermal growth factor receptor staining (for interleukin-6 r = 0.29; 95% CI 0.07, 0.51; p = 0.01 and for cyclic guanosine monophosphate r = 0.34; 95% CI 0.13, 0.55; p = 0.002). Urine interleukin-8 was negatively associated with urothelial heparin-binding epidermal growth factor-like growth factor staining (r = -0.34; 95% CI -0.55, -0.12; p = 0.002) and positively associated with lamina propria mast cell count (r = 0.29; 95% CI 0.06, 0.52; p = 0.01). The latter association also was seen in treated patients (r = 0.46; 95% CI 0.20, 0.73; p <0.001). None of the urine markers was significantly different for ulcer vs nonulcer groups. All of the patients with ulcer had extensive inflammation on bladder biopsy including severe mononuclear cell infiltration, moderate or strong interleukin-6 staining in the urothelium and lamina propria, and leukocyte common antigen staining in more than 10% of the lamina propria. However, these features also were seen in 24% to 76% of the patients without ulcer. CONCLUSIONS: Overall urine markers did not associate robustly with biopsy findings. The strongest association was a positive association between urine interleukin-8 levels and bladder mast cell count. Patients with ulcer consistently had bladder inflammation but the cystoscopic finding of ulcers was not a sensitive indicator of inflammation on bladder biopsy.

Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method.

Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.

Toxicokinetic study of recombinant human heparin-binding epidermal growth factor-like growth factor (rhHB-EGF) in female Sprague Dawley rats.

PURPOSE: To determine the toxicity and pharmacokinetics of recombinant heparin-binding epidermal growth factor-like growth factor in female Sprague Dawley rats following intra-bladder and intravenous administration. MATERIALS AND METHODS: rhHB-EGF was administered once daily for 6 or 27 days at doses of 3, 10, or 30 microg/kg. 125I-rhHB-EGF was administered on day 7 or 28 for pharmacokinetic analysis. Toxicity was assessed by general appearance and behavior, gross necropsy, blood chemistry and microscopic evaluation. RESULTS: Plasma AUCss of [125I] rhHB-EGF equivalents following IB administration for 7 days were 4.28+/-2.29, 7.75+/-2.70, and 7.11+/-1.42 ng ml(-1) h(-1) at doses of 3, 10, and 30 microg/kg, respectively. Following IV administration, the AUCss on day 7 increased from 27.0+/-2.66 to 124+/-5.09 and 385.11+/-7.57 ng ml(-1) h(-1) with increasing the dose from 3 to 10 and 30 microg/kg. Similar AUCss data was obtained after 28 day administration. No toxicity was evident upon gross examination. Histologic examination revealed subacute inflammation and lymphocytic infiltration of the urinary bladder in animals from all groups dosed by the IB route. CONCLUSIONS: Plasma and bladder concentrations of recombinant human [125I] rhHB-EGF equivalents were significantly lower following the IB route than following IV administration. Histologic tissue examination indicated no toxicity attributable to rhHB-EGF.

p53 mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells.

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis, a urinary bladder disorder of unknown etiology that is characterized by chronic pelvic pain. The present study was directed toward uncovering a pathway through which APF signals. Treatment of human urothelial cells with native APF resulted in growth inhibition accompanied by blockade of cell cycle transit and increased p53. Reduced expression of p53 by RNA interference diminished, while ectopic expression of p53 mimicked, the effects of APF. These are the first findings implicating the network of p53 target genes in urothelial defects associated with interstitial cystitis.