Transcriptome profiling of bovine ovarian theca cells treated with fibroblast growth factor 9.

We reported previously that fibroblast growth factor 9 (FGF9) acts as an antidifferentiation factor, stimulating proliferation of granulosa cells (GCs) and theca cells (TCs) while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TCs were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. After treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cutoff, and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements upregulated and 191 elements downregulated by FGF9. The ingenuity pathway analysis (IPA) was used to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions and unveiled interesting relationships among genes related to cell proliferation (eg, CCND1, FZD5, and MYB), antioxidation/cytoprotection (eg, HMOX1 and NQO1), and steroidogenesis (eg, CYP11A1 and STAR). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development.

Assessment of colon and bladder crosstalk in an experimental colitis model using contrast-enhanced magnetic resonance imaging.

BACKGROUND: Inflammatory bowel disease (IBD) consists of two chronic remitting-relapsing inflammatory disorders in the colon referred to as ulcerative colitis and Crohn's disease (CD). Inflammatory bowel disease affects about 1.4 million Americans. 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis is a widely used model of experimental intestinal inflammation with characteristic transmural and segmental lesions that are similar to CD. METHODS: Here, we report on the use of contrast-enhanced magnetic resonance imaging (CE-MRI) to monitor in vivo bladder permeability changes resulting from bladder crosstalk following colon TNBS exposure, and TNBS-induced colitis. Changes in MRI signal intensities and histology were evaluated for both colon and bladder regions. KEY RESULTS: Uptake of contrast agent in the colon demonstrated a significant increase in signal intensity (SI) for TNBS-exposed rats (p < 0.01) compared to controls. In addition, a significant increase in bladder SI for colon TNBS-exposed rats (p < 0.001) was observed compared to saline controls. Histological damage within the colon was observed, however, bladder histology indicated a normal urothelium in rats with TNBS-induced colitis, despite increased permeability seen by CE-MRI. CONCLUSIONS & INFERENCES: Contrast-enhanced MRI was able to quantitatively measure inflammation associated with TNBS-induced colitis, and assess bladder crosstalk measured as an increase in urothelial permeability. Although CE-MRI is routinely used to assess inflammation with IBD, currently there is no diagnostic test to assess bladder crosstalk with this disease, and our developed method may be useful in providing crosstalk information between organ and tissue systems in IBD patients, in addition to colitis.

Template-driven gene selection procedure.

The hierarchical clustering and statistical techniques usually used to analyse microarray data do not inherently represent the underlying biology. Herein, a hybrid approach involving characteristics of both supervised and unsupervised learning is presented. This approach is based on template matching in which the interaction of the variables of inherent malignancy and the ability to express the malignant phenotype are modelled. Immortalised normal urothelial cells and bladder cancer cells of different malignancy were grown in conventional two-dimensional tissue culture and in three dimensions on extracellular matrices (ECMs) that were either permissive or restrictive for expression of the malignant phenotype. The transcriptome represents the effects of two variables--inherent malignancy and the modulatory effect of ECM. By assigning values to each of the biological variables of inherent malignancy and the ability to express the malignant phenotype, a template was constructed, which encapsulated the interaction between them. Gene expression correlating both positively and negatively with the template was observed, but when iterative correlations were carried out, the different models for the template converged on the same actual template. A subset of 21 genes was identified, which correlated with two a priori models or an optimised model above the 95% confidence limits identified in a bootstrap resampling with 5000 permutations of the data set. The correlation coefficients of expression of several genes were > 0.8. Analysis of upstream transcriptional regulatory elements (TREs) confirmed that these genes were not a randomly selected set of genes. Several TREs were identified as significantly over-expressed in the sample of 20 genes for which TREs were identified, and the high correlations of several genes were consistent with transcriptional co-regulation. The authors suggest that the template method can be used to identify a unique set of genes for further investigation.

Biomarker risk assessment and bladder cancer detection in a cohort exposed to benzidine.

BACKGROUND: Cancer screening with highly sensitive, specific biomarkers that reflect molecular phenotypic alterations is an attractive strategy for cancer control. We examined whether biomarker profiles could be used for risk assessment and cancer detection in a cohort of Chinese workers occupationally exposed to benzidine and at risk for bladder cancer. METHODS: The cohort consisted of 1788 exposed and 373 nonexposed workers, followed from 1991 through 1997. We assayed urothelial cells from voided urine samples for DNA ploidy (expressed as the 5C-exceeding rate [DNA 5CER]), the bladder tumor-associated antigen p300, and a cytoskeletal protein (G-actin). Workers were stratified into different risk groups (high, moderate, and low risk) at each examination based on a predefined biomarker profile. For workers who developed bladder cancer, tumor risk assessment was analyzed from samples collected 6-12 months before the cancer diagnosis. The associations between risk group and subsequent development of bladder cancer were analyzed by Cox proportional hazards regression analysis and logistic analysis, after adjustment. All statistical tests were two-sided. RESULTS: Twenty-eight bladder cancers were diagnosed in exposed workers and two in nonexposed workers. For risk assessment, DNA 5CER had 87.5% sensitivity, 86.5% specificity, an odds ratio (OR) of 46.2 (95% confidence interval [CI] = 8.1 to 867.0), and a risk ratio (RR) of 16.2 (95% CI = 7.1 to 37.0); p300 had 50.0% sensitivity, 97.9% specificity, an OR of 40.0 (95% CI = 9.0 to 177.8), and an RR of 37.9 (95% CI = 16.8 to 85.3). The risk of developing bladder cancer was 19.6 (95% CI = 8.0 to 47.9) times higher in workers positive for either the DNA 5CER or p300 biomarkers than in workers negative for both biomarkers and 81.4 (95% CI = 33.3 to 199.3) times higher in workers positive for both biomarkers. G-actin was a poor marker of individual risk. CONCLUSIONS: Occupationally exposed workers at risk for bladder cancer can be individually stratified, screened, monitored, and diagnosed based on predefined molecular biomarker profiles.

Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy.

Because small intestine submucosa (SIS) is a bioscaffold for tissue regeneration, we describe a method to analyze the material for growth peptides and for structural molecules. Immunofluorescence methods are described for relative quantification of abundant structural proteins. Additionally, a quantitative technique for comparison of the content of less abundant proteins in SIS was developed using the tyramide signal amplification (TSA) system that is applicable to paraffin-preserved tissue blocks. Frozen sections generally shredded when cut thinly enough to permit entry and washout of reagents. Five micrometer sections cut from paraffin blocks were immunolabeled for collagen, heparan sulfate proteoglycans (HSPG), FGF2, TGFbeta, and VEGF. Images of tissue sections were acquired by a linear image camera and quantified by densitometry after thresholding the signal to minimize nonspecific fluorescence. Immunohistochemistry was used to confirm the immunofluorescence methods. HSPG was widely distributed but concentrated in vessels. FGF2 was distributed diffusely and was associated with fibrous structures. VEGF was distributed mainly around vessels. TGFbeta was barely detectable above background. Collagen fibrils were distinctly present, and with a two-color fluorescence system, the distribution of components relative to collagen can be assessed. The anatomic structure of SIS is likely to play an important role in the regeneration of tissues, and factors in remnant vessels may facilitate penetration of the matrix along these avenues.

Abnormal G-actin content in single prostate cells as a biomarker of prostate cancer.

The distribution of altered G-actin was investigated in prostatic cells obtained by fine needle aspiration (FNA) from 27 excised prostate glands obtained during radical prostatectomy. FNA, which was used to obtain single cells for image analysis, sampled in the region of any nodules and in grossly normal areas of the contralateral lobes. Quantitative fluorescence-image analysis was used to assay the amount of G-actin in individual cells. Abnormal G-actin, a precursor cytoskeletal protein representing cytoskeletal rearrangements accompanying cellular transformation, was associated with the presence of adenocarcinoma in 22 of 27 specimens from the dominant nodule, but only 3 of 20 in the grossly normal specimens (P<.0001). The mean G-actin content of all samples from the dominant nodule was 113.2+/-6.87 and 69.57+/-4.47 from the grossly normal area, the difference being significant at P<.0001. Altered G-actin was not associated with Gleason score (P = .95), grade (P = .26), stage (P = .058), or tumor volume (P = .32), thereby indicating it is a general marker for prostate adenocarcinoma.

Loss of tissue transglutaminase as a biomarker for prostate adenocarcinoma.

BACKGROUND: Additional molecular tissue biomarkers for prostate carcinoma are needed to stratify patients with clinically suspicious findings, such as an elevated prostate specific antigen (PSA) with a negative biopsy, according to risk. METHODS: Prostate tissues from 43 cancer cases and 47 controls with no evidence of cancer were labeled for transglutaminase by immunohistochemistry. Immunoreactivity was quantified using the Autocyte Pathology Workstation. In addition, quantitative fluorescence image analysis was used to compare transglutaminase concentrations in cells obtained by fine-needle aspiration from excised prostates. Loss of gene expression was evaluated by reverse transcriptase-polymerase chain reaction and growth with 5-azacytidine. RESULTS: Visually, benign glands from controls generally expressed tissue transglutaminase, whereas regions with adenocarcinoma generally were negative. With quantitative immunohistochemistry, 41 of 43 adenocarcinoma of the prostate (CaP) cases expressed lower mean percentage areas positive for transglutaminase than did 30 of 30 benign prostatic hyperplasia (BPH) and 17 of 17 prostatitis cases (P < 0.0001; odds ratio [OR], 1577; 95% confidence interval (CI), 74-33, 820; relative risk [RR], 25; 95% CI, 6-95). Quantitative immunofluorescence of 3277 cells collected by FNA from 19 CaP cases and 645 cells from 5 cases of BPH showed that the mean content of transglutaminase was 93 femtograms (fg) for the CaP-derived cells and 138 fg for the BPH cells (P < 0.0001). Receiver operating curve analysis of the immunohistochemistry data showed an optimized threshold produced 95% sensitivity with 100% specificity. Growth of LNCaP cells with 5-azacytidine failed to stimulate transglutaminase expression, suggesting that loss of expression was likely not attributable to promoter methylation. CONCLUSIONS: Measurements of transglutaminase on tissue sections provides additional diagnostic information that is potentially useful for risk assessment of patients with suspicious clinical findings, such as nodules or positive PSA and negative biopsies, without overdetecting disease.

Bexarotene ligand pharmaceuticals.

Bexarotene (LGD-1069), from Ligand, was the first retinoid X receptor (RXR)-selective, antitumor retinoid to enter clinical trials. The company launched the drug for the treatment of cutaneous T-cell lymphoma (CTCL), as Targretin capsules, in the US in January 2000 [359023]. The company filed an NDA for Targretin capsules in June 1999, and for topical gel in December 1999 [329011], [349982] specifically for once-daily oral administration for the treatment of patients with early-stage CTCL who have not tolerated other therapies, patients with refractory or persistent early stage CTCL and patients with refractory advanced stage CTCL. The FDA approved Targretin capsules at the end of December 1999 for once-daily oral treatment of all stages of CTCL in patients refractory to at least one prior systemic therapy, at an initial dose of 300 mg/m2/day. After an NDA was submitted in December 1999 for Targretin gel, the drug received Priority Review status for use as a treatment of cutaneous lesions in patients with stage IA, IB or IIA CTCL [354836]. The FDA issued an approvable letter in June 2000, and granted marketing clearance for CTCL in the same month [370687], [372768], [372769], [373279]. Ligand had received Orphan Drug designation for this indication [329011]. At the request of the FDA, Ligand agreed to carry out certain post-approval phase IV and pharmacokinetic studies [351604]. The company filed an MAA with the EMEA for Targretin Capsules to treat lymphoma in November 1999 [348944]. The NDA for Targretin gel is based on a multicenter phase III trial that was conducted in the US, Canada, Europe and Australia involving 50 patients and a multicenter phase I/II clinical program involving 67 patients. Targretin gel was evaluated for the treatment of patients with early stage CTCL (IA-IIA) who were refractory to, intolerant to, or reached a response plateau for at least 6 months on at least two prior therapies. Efficacy results exceeded the protocol-defined response target rates; side effects were primarily limited to local skin reactions [349982]. Ligand has worldwide rights to market bexarotene capsules, and will market the drug in the US, Canada and selected European markets. In Spain, Portugal, Greece and Central and South America, Ferrer Internacional will market and distribute the drug. As of December 1999, Ligand was seeking additional distribution partners for select European and Asian markets [351604]. In January 2000, Alfa Wassermann signed an agreement with Ligand to exclusively market and distribute Targretin gel and capsules in Italy. Alfa paid US $0.75 million on signing with additional amounts up to an aggregate total of US $1.0 million on achievement of certain registration milestones, which are expected to be met in 2000 [351882].

Complexity, retinoid-responsive gene networks, and bladder carcinogenesis.

Carcinogenesis involves inactivation or subversion of the normal controls of proliferation, differentiation, and apoptosis. However, these controls are robust, redundant, and interlinked at the gene expression levels, regulation of mRNA lifetimes, transcription, and recycling of proteins. One of the central systems of control of proliferation, differentiation and apoptosis is retinoid signaling. The hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network. In this study we showed that simple, cause-effect interpretations in terms of hRAR alpha gene transcription being the central regulatory event would not describe the retinoid-responsive gene network. A set of cultured bladder-derived cells representing different stages of bladder tumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers. Relative gene expression before (constitutive) and after treatment with 10 microM all-trans-retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, hRAR alpha nd beta, hRXR alpha and beta CRBP, CRABP I and II; and for signaling genes that are known to be sensitive to retinoic acid, EGFR, cytokine MK, ICAM I and transglutaminase. The phenotype for inhibition of proliferation and for apoptotic response to both aTRA and the synthetic retinoid 4-HPR was determined. Transfection with a CAT-containing plasmid containing an aTRA-sensitive promoter was used to determine if the common retinoic acid responsive element (RARE)-dependent pathway for retinoid regulation of gene expression was active. Each of the genes selected is known from previous studies to react to aTRA in a certain way, either by up- or down-regulation of the message and protein. A complex data set not readily interpretable by simple cause and effect was observed. While all cell lines expressed high levels of the mRNAs for hRXR alpha and beta that were not altered by treatment with exogenous aTRA, constitutive and stimulated responses of the other genes varied widely among the cell lines. For example, CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively. The expression of hRAR alpha, which governs the expression of many retinoid-sensitive genes, was expressed at moderate to high levels in all cell lines, but in some it was sharply upregulated (TCC-Sup, HUC-PC and J82), remained constant (5637 and HUC-BC), or was down-regulated (SCaBER, T24 and RT4). The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa. One line (RT4) was insensitive to either retinoid. Transfection showed very little retinoid-stimulated transfection of the CAT reporter gene with RT4 or HUC-PC. About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3-8 fold with 5637, TCC-Sup cells. In HUC-BC, a G to T point mutation was found at position 606 of the hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain. These data indicate that retinoid signaling is probably a frequent target of inactivation in bladder carcinogenesis. (ABSTRAC

Retinoid signaling in immortalized and carcinoma-derived human uroepithelial cells.

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 microM of either all-trans or 13-cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.

Biomarkers in monitoring for efficacy of immunotherapy and chemoprevention of bladder cancer with dimethylsulfoxide.

This study correlated biomarkers expressed in tumor and epithelial field with clinical response and recurrence. Of 25 bladder cancer patients, 11 received 6 weeks of intravesical Bacille Calmette-Guerin (BCG), and 14 were treated weekly with intravesical dimethylsulfoxide (DMSO) for 4 weeks to further modulate biomarker expression. G-actin, DNA aneuploidy, and p300 tumor antigen were evaluated by quantitative fluorescence image analysis on uroepithelial cells from bladder wash samples prior to and immediately following treatment. Excluding patients who did not respond to BCG (and who had persistently abnormal p300 and DNA markers), recurrence correlated with persistent abnormal G-actin findings. Of patients who were G-actin negative following therapy, only 25% recurred during follow-up in contrast to 67% in patients who were positive (p < 0.03 by Fisher's exact test). The odds ratio for recurrence was 6.00 (95% confidence interval: 1.3-28.6). Cytosolic G-actin levels can be an important intermediate end point marker for chemoprevention.

Expression of retinoid-responsive genes occurs in colorectal carcinoma-derived cells irrespective of the presence of resistance to all-trans retinoic acid.

BACKGROUND AND OBJECTIVES: Retinoids are metabolized in human intestinal epithelial cells to all-trans retinoic acid; however, it is unknown whether these cells express retinoid receptors, and whether sensitivity or resistance to the hormone is associated with a particular pattern of expression of retinoid-responsive genes. METHODS: Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were used to identify mRNAs for retinoid receptors. Both Relative RT-PCR and transfection of retinoid-inducible plasmid were applied to test functionality of the pathway in a model system for colorectal carcinoma progression (primary SW480, all-trans retinoic acid-sensitive cells vs. metastatic SW620, -insensitive cells). RESULTS: Three colorectal carcinoma-derived cell lines were inhibited by the hormone. Retinoic acid receptor type alpha (hRAR alpha) and retinoid X receptor type alpha (hRXR alpha) mRNAs were detected in normal enterocytes, colonocytes, and in all colorectal carcinoma-derived cells studied. Primary carcinomas and metastatic lesions expressed high amounts of hRAR alpha receptor protein, showing no simple correlation between the amounts of mRNA and receptor protein. No pattern of expression of the retinoid-responsive genes was associated with sensitivity or resistance to the retinoid. Expression of the genes occurred irrespective of resistance to the hormone or inactivity of the pathway. CONCLUSIONS: Colonocytes possess a molecular system for transduction of the retinoid signal. All-trans retinoic acid modifies gene expression and inhibits proliferation of these cells. Therefore, retinoids are likely to be effective in chemoprevention of colorectal carcinoma.

Quantitative changes in cytoskeletal and nuclear actins during cellular transformation.

Actin, a highly conserved protein comprising cell stress fibers and other cellular structures, is found in both the cytoplasm and nucleus of cells and responds to both epigenetic signals and altered gene expression occurring during tumorigenesis. We have previously shown that changes in the cytoplasmic F- and G-actin ratios reflect bladder cancer risk. To determine whether nuclear actin is also altered and how nuclear and cytoplasmic actin alterations are interrelated in transformation, an in vitro model of carcinogen-induced transformation consisting of 2 human uroepithelial cell lines immortalized by infection with SV-40 was studied. One line, HUC-PC, is tumorigenic in nude mice after incubation with the carcinogen 4-ABP, the other, HUC-BC, is not. Cytoplasmic and nuclear F- and G-actin were determined by QFIA on individual cells using fluorochrome-labeled phallicidin and DNase, I, respectively. Before exposure to 4-ABP, the PC cells had lower cytoplasmic F-actin content, higher cytoplasmic G-actin content, but similar levels of nuclear G- and F-actin in comparison to the BC cells. After incubation with 4-ABP, F-actin decreased and G-actin increased in both cytoplasm and nuclei of PC cells and cytoplasmic F-actin fibers were lost, but only cytoplasmic actin was altered in the BC cells. Northern blot analysis showed the expression of the beta-actin gene was only approximately 20% lower in 4-ABP-treated PC cells than in untreated controls, indicating the cellular change in actin was attributed to a shift between F- and G-actin proteins rather than to net actin synthesis.

Molecular study of sex steroid receptor gene expression in human colon and in colorectal carcinomas.

BACKGROUND: Sex steroid hormones influence function of the human gastrointestinal tract. Although the specific receptor proteins have been identified in surgical specimens of both intestinal mucosa and colorectal carcinomas, it is still unknown whether they are expressed in intestinal epithelial cells. METHODS: Expression of androgen receptor (AR) protein and estrogen receptor (ER) protein was studied by Scatchard analysis and ELISA (for ER only) in surgical specimens of normal-appearing mucosa, colorectal carcinomas, isolated colonocytes, and human colorectal carcinoma cell lines. Northern analysis was applied to identify the appropriate mRNAs, followed by the sensitive technique of reverse transcription-polymerase-chain-reaction (RT-PCR). RESULTS: AR protein was identified in all surgical specimens analyzed and ER protein in 10 out of 13 normal-appearing mucosa specimens and 4 out of 7 colorectal carcinomas. The receptor proteins were not found in isolated colonocytes or in the transformed cell lines. RT-PCR confirmed that none of the isolated normal colonocytes or transformed colorectal carcinoma-derived cells expressed these mRNAs. Intestinal smooth muscle cells and fibroblasts were found to express sex steroid receptor mRNAs. CONCLUSIONS: Both receptors are present in human large intestine but are expressed in stromal cells and not in intestinal epithelial cells. We hypothesize that sex steroids may influence the function of colonocytes indirectly through stromal-epithelial interactions.

Neural net-based identification of cells expressing the p300 tumor-related antigen using fluorescence image analysis.

We report on preliminary investigations of the use of an image analysis system to perform preliminary algorithmic classification of images of fluorochrome-labeled cells followed by capture of gray-level images of potentially abnormal cells for analysis by a neural network. Cells were labeled with an antibody against a bladder cancer tumor-associated antigen, and the neural net was used to distinguish true-positive cells from negative cells, false-positive cells (autofluorescent or nonspecific labeling), and cell-sized artifacts. Gray-level cell images were digitized and processed for analysis by a feed-forward neural network using back-propagation. The network was trained and tested with two independent image sets. Various network configurations and activation functions were investigated, including a sinusoidal activation function. At high power, the network agreed completely with the human observer's classification. At low power, a strong clustering of cells classified by the network with expert classification was seen, while the neural network showed roughly 75% concordance with the human observer. In addition, a set of four features extracted from raw cell images were investigated. The features were: shape factor, texture, area, and average pixel intensity. A network trained with these features performed better than one operating with gray-level images. We conclude that using neural networks to recognize and classify images captured by an image analysis microscope is feasible.

Expression of sex steroid receptor genes and comodulation with retinoid signaling in normal human uroepithelial cells and bladder cancer cell lines.

The expression of sex steroid receptor genes in human uroepithelial cells (UEC) and their role in bladder carcinogenesis is unknown. Expression of androgen receptor (hAR), estrogen receptor (hER), and vitamin d3 receptor (hVDR3) genes in normal human stromal cells (SC) and UEC, six bladder cancer cell lines, and two SV-40-immortalized cell lines (SVC) was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Functionality was assessed indirectly by relative RT-PCR, which identified comodulation of mRNA expression between retinoic acid and sex steroid receptor genes. UEC and SC expressed hAR and hER mRNA constitutively at low levels, but only positive controls expressed hVDR3. Every cancer cell line and the SVC showed aberrant expression. Treatment of cells with all-trans-retinoic acid up-regulated hAR and hER expression, whereas treatment with sex steroids up-regulated retinoic acid receptor expression. Cell proliferation was not affected by sex steroids or by their inhibitors. Sex steroid signaling pathways are functional in UEC and appear to be altered during bladder tumorigenesis. The sex steroid receptors may play a role in normal differentiation.

Characterization of the DD23 tumor-associated antigen for bladder cancer detection and recurrence monitoring. Marker Network for Bladder Cancer.

Bladder cancer detection, monitoring, and prevention represent major problems that could be addressed with sensitive and specific biomarkers. The antigen recognized by the DD23 antibody, previously developed against a tumor-related antigen, was partially biochemically characterized, and its sensitivity and specificity in cancer detection and recurrence monitoring was evaluated. Quantitative fluorescence image analysis was used to quantify antigen content in exfoliated urothelial cells in a cross-section of patients with bladder cancers of all grades and stages and control populations. The antigen was found in tumor cells as well as normal-appearing urothelial cells, suggesting it represents a marker induced by the altered growth factor environment of a cancer-containing bladder. When used as a quantitative marker, the sensitivity for bladder cancer detection was 85%, and the specificity was 95%. No significant difference was seen between symptomatic and asymptomatic control populations, including patients with previous bladder cancers in the absence of a recurrence. In bladder cancer recurrence monitoring, results were consistently negative until just before detection of a recurrence. The biomarker reflects a "field effect" that occurs very late in tumorigenesis and seems to represent events common to most cancers involving the genitourinary tract. Western blotting showed the antibody recognized a dimeric protein. DD23 quantification in single cells may be particularly useful in targeting cystoscopic intervention for recurrence monitoring and, because of its high specificity, could be a tool for bladder cancer screening in high-risk groups.

A deficit of chondroitin sulfate proteoglycans on the bladder uroepithelium in interstitial cystitis.

OBJECTIVES: To investigate the abundance of chondroitin sulfate proteoglycans at the bladder lumenal and subepithelial surfaces in bladder biopsies derived from patients with interstitial cystitis (IC) and controls. METHODS: Tissue sections derived from biopsies from 31 IC patients and 24 pathologically normal control sections were labeled for proteoglycans using the 2B6 anti-"stub" antibody and detected by immunohistochemistry. RESULTS: On the lumenal surface, 5 of 31 (19%) IC sections were positive for proteoglycans versus 14 of 24 (58%) control sections (P = 0.00011). At the basal surface, 5 of 19 IC patients were positive versus 7 of 12 controls (P = 0.032). CONCLUSIONS: A deficit of bladder lumenal and basal proteoglycans is associated with IC. The deficit in basal layer proteoglycans suggests an altered urothelial differentiation program. The lumenal deficit suggests that the charge-dependent exclusion of ions from the bladder surface is compromised in IC.