NuMA: evaluation of a new biomarker for the detection of low stage colorectal cancer.

An enzyme immunoassay for NuMA was evaluated in a retrospective clinical study for its potential utility in the detection of colorectal cancer. The concentrations of NuMA and CEA (Abbott IMx) were measured in sera from 86 patients (presurgical) with colorectal cancer, 72 subjects with benign gastrointestinal diseases, 80 subjects with risk factors for colorectal cancer, and 141 age-matched healthy subjects. Reference values for NuMA and CEA were calculated by two methods: 95% cumulative distribution and ROC analyses versus healthy subjects. By the first method, NuMA and CEA both had approximately 20% sensitivity for colorectal cancer. By the second method (which generated lower reference values), NuMA was more sensitive than CEA for colorectal cancer. This improved sensitivity was most evident in Dukes B subjects. By either analysis method, NuMA was more sensitive than CEA for subjects at risk for developing colorectal cancer, whereas CEA was more specific for benign gastrointestinal diseases.

Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4-216) recognizes nucleophosmin/B23.

BACKGROUND: The nuclear protein B23, nucleophosmin, is an RNA-associated nucleolar phosphoprotein reported to be more abundant in malignant and growing cells than in normal nondividing cells. We examined the levels of B23 in fresh human prostate tissue and in five human prostate cancer cell lines with monoclonal antibodies (mAb) to nucleophosmin (alpha-B23) and to human prostate cancer nuclear matrix proteins (PRO:4-216). METHODS: mAb PRO:4-216 and mAb alpha-B23 were used for protein level detection. Nuclear matrix proteins (NMPs) were prepared from prostate tumor and five human prostate cancer cell lines: LNCaP, TSU, DU145, PC-3, and PPC-1. The NMPs were run on one-dimensional and two-dimensional (2D) electrophoresis gels for Western blot analysis with the two mAbs. Histologic sections from paraffin-embedded normal and cancerous prostate tissue were stained immunohistochemically with both mAbs. RESULTS: PRO:4-216 and B23 mAbs identified a 40-kD protein (pI approximately 5.0) by Western blot analysis in the human prostate cancer cell lines and on two-dimensional blots of human prostate cancer NMPs. Immunohistochemical staining demonstrated large punctate nuclear dots in most cancer nuclei, while staining of normal tissue was less intense or absent. Predominant reactivity was of epithelial nuclei, with some minor reactivity of stromal nuclei. Red blood cells (RBCs) and white blood cells (WBCs) were routinely negative. CONCLUSIONS: PRO:4-216, previously characterized as recognizing prostate cancer nuclear matrix proteins, recognized B23/nucleophosmin. PRO:4-216 and alpha-B23 showed intense immunohistochemical staining of B23/nucleophosmin in cancer nuclei compared to adjacent normal cells in paraffin-embedded prostate tissue. This preliminary study showed the potential of B23 as a tumor marker for human prostate cancer.

Preliminary immunohistochemical characterization of a monoclonal antibody (PRO:4-216) prepared from human prostate cancer nuclear matrix proteins.

OBJECTIVES: A nuclear matrix protein (PC-1) was previously identified and reported to be present only in human prostate cancer but absent in tissue from the same prostate containing either benign prostatic hyperplasia (BPH) or normal prostate tissue. The PC-1 protein was identified by high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and exhibited a molecular mass of 56 kDa and an isoelectric point of 6.58. This work investigates the immunohistochemical characterization of PRO:4-216, a monoclonal antibody to PC-1. METHODS: Areas of the 2D-PAGE gels containing the human prostate cancer nuclear matrix proteins near PC-1 were isolated, eluted, and injected into mice to develop monoclonal antibodies. Antibodies were screened by immunofluorescence for nuclear reactivity to a human prostate cancer cell line (LnCaP) and by 1D and 2D Western blots for reactivity with prostate cancer nuclear matrix proteins. Monoclonal antibodies from the selected clones were affinity purified. The monoclonal antibody PRO:4-216 was used to analyze frozen tissue from 20 cancerous, 22 BPH, and 22 normal regions from fresh human prostate specimens. Tissue sections were analyzed for their immunohistochemical (IHC) (horseradish peroxidase) staining. RESULTS: Using a reference value for positive staining at an IHC score of greater than 50, 85% (17 of 20) of the cancerous, 5% (1 of 22) of the BPH, and 9% (2 of 22) of the normal prostate tissues stained positive. The one BPH and two normal tissues that stained positive were taken from prostates in which the adjacent cancerous tissue also demonstrated high IHC scores (greater than 225). CONCLUSIONS: These data demonstrate nuclear reactivity on fresh frozen human prostate cancer tissue for the monoclonal antibody PRO:4-216. PRO:4-216 may aid in distinguishing normal prostate and BPH from cancerous tissue.

Urinary nuclear matrix protein as a marker for transitional cell carcinoma of the urinary tract.

PURPOSE: The purpose of this trial was to evaluate an immunoassay for urinary nuclear matrix protein, NMP22, as an indicator for transitional cell carcinoma of the urinary tract. MATERIALS AND METHODS: Three groups of subjects participated in this trial of NMP22: 1-175 with transitional cell carcinoma, 2-117 with benign urinary tract conditions and 3-375 healthy volunteers. Each subject provided a single (3 voids) urine sample for analysis at the time of study entry. Each sample was assayed for the level of NMP22. RESULTS: In normal healthy volunteers and in subjects with benign conditions median NMP22 levels were 2.9 and 3.3 units per ml., respectively. Median urinary NMP22 levels in patients with transitional cell carcinoma were significantly greater than in comparison subjects. Patients with active transitional cell carcinoma had significantly greater median urinary NMP22 levels than those with no evidence of disease (6.04 versus 4.11 units per ml., p = 0.027, 1-tailed Mann-Whitney U test). We noted no effect of tumor grade, extent of disease or exposure to intravesical therapy on urinary NMP22 levels. CONCLUSIONS: NMP22 is a promising urinary tumor marker for monitoring transitional cell carcinoma. Nuclear matrix proteins are a new class of tumor markers that represent the basis for the development of assays with increased efficacy for the detection and treatment of cancer.

Utilization of nuclear matrix proteins for cancer diagnosis.

Circulating tumor markers have been used increasingly in recent years as clinical tools for cancer diagnosis and management. This review presents a brief discussion of currently available tumor-associated antigens. Included is an overview of different functional classes of circulating markers and their clinical applications. The limitations of some traditional tumor markers presently in widespread use are discussed in the context of the properties exhibited by an ideal tumor marker. The nuclear matrix provides structural support for the nucleus and plays a dynamic role in the spatial organization of the genome and in the control of DNA replication and transcription. The recovery of increased amounts of specific nuclear matrix proteins in several different cancers has led to the further study of some of these proteins as a new class of tumor markers. Progress on the use of a nuclear matrix protein known as NuMA as a marker for bladder cancer is presented, including results of a recently completed multisite clinical trial. Additional studies on the potential utility of nuclear matrix proteins as markers for prostate cancer are also presented. Nuclear matrix proteins could provide for the development of assays with increased efficacy for the diagnosis and treatment of cancer.

An enzyme immunoassay for carcinoembryonic antigen which employs simultaneous incubation of specimen with solid phase and enzyme-conjugated antibodies.

The Abbott CEA-EIA Monoclonal One-Step procedure was evaluated and compared to the Abbott CEA-EIA Monoclonal, a two-step assay. The reproducibility, sensitivity, recovery and dilution linearity were similar for both assays. Excellent correlations were found between the carcinoembryonic antigen (CEA) values obtained with these assays for healthy donors (n = 261, r = 0.951), patients with benign diseases (n = 171, r = 0.994) and cancer patients (n = 585, r = 0.997). In serial monitoring studies, one-step CEA values paralleled the CEA values determined with the two-step assay regardless of the type of cancer. The advantages of the one-step CEA assay over the two-step assay include significant reduction in both attended and total assay times, reduced sample volume, dry antibody-coated beads, color-coded reagents and an extended range for the standard curve. These test improvements were achieved without affecting the reproducibility, sensitivity and accuracy of the CEA measurement.

Tubulocisternal endoplasmic reticulum in human eccrine sweat glands.

Human eccrine sweat glands, including those expressing the gene for cystic fibrosis, were stained sequentially en bloc with uranyl acetate, lead citrate and copper sulfate, and osmium tetroxide for the ultrastructural demonstration of tubulocisternal endoplasmic reticulum (TER). This organelle has been associated with water and electrolyte transport in other epithelia. TER was present in the apical cytoplasm and basolateral cytoplasm circumscribing intercellular canaliculi of clear cells in the secretory coil and in the apical cuticular cytoplasm of luminal cells in the coiled duct. In both the secretory coil and duct, TER was situated strategically around the tight junctions. Together, the TER and tight junctions may regulate the secretory and absorptive permeability of the eccrine sweat gland to NaCl and water. The dark cell of the coil which is exocrine, and the deep cell of the duct which lacked tight junctions, did not contain TER. In sweat glands obtained from three cases of cystic fibrosis, elements of TER were more prevalent and complex in luminal cells of the secretory duct than was the TER in normal sweat glands. This difference may be a consequence of the basic electrolyte abnormality of cystic fibrosis.

Immunohistochemical localization of carbonic anhydrase I and II in eccrine sweat glands from control subjects and patients with cystic fibrosis.

Isozymes of carbonic anhydrase (CA) were localized immunohistochemically by the immunoglobulin-peroxidase bridge technique on fixed paraffin sections of human eccrine sweat glands. Low-activity CA I was identified in the cytoplasm of the myoepithelial cells in the secretory coil and in the luminal and basal cells of both the coiled and straight segments of the duct. High-activity CA II was found in the cytoplasm of clear cells of the secretory coil. Although evidence has suggested that CA activity is altered in cystic fibrosis (CF), the present immunohistochemical comparison of CF sweat glands revealed a distribution of and, semiquantitatively, a prevalence of CA isozymes identical to those of normal sweat glands. Abnormal enzyme activity cannot be ruled out, however, on the basis of immunocytochemical staining which depends solely on the antigenic properties of CA.

Freeze fracture morphology of the tight junctions of the eccrine sweat gland from patients with cystic fibrosis.

The resorption of Na+ and Cl- across the duct of the human eccrine sweat gland is markedly decreased in individuals with cystic fibrosis (CF). Conceivably, a defective transcellular ion transport mechanism or an increased paracellular backflux of ions could account for the abnormal salt resorption in the sweat gland duct and other organs affected in CF. Tight junctions are thought to regulate paracellular ion flow. Specifically, the number of junctional elements observed by freeze fracture are believed to correspond with the extent of paracellular transport. We compared the freeze fracture morphology of tight junctions of eccrine sweat glands taken from 11 control and seven CF patients. In an attempt to "fingerprint" the junctions morphometrically, the following parameters were measured: the number of strands, the depth of the junction from the apical to the basal strand, the angle of intersection between strands, and the mean distance along a strand between intersections with two other strands. No significant difference was observed between control and CF sweat glands in the freeze fracture morphology of the tight junctions of the duct, the segment where the net reabsorption of Na+ and Cl- is abnormally decreased in CF. Significant changes were observed, however, in the means of the number of strands, the depth, and the distance between intersections for the tight junctions of the intercellular canaliculus of the secretory coil, which appears to function normally in CF.

A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes.

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.

The intracellular localization of amiloride in frog skin.

The diuretic compound amiloride is often used as a specific inhibitor of the passive Na+ entry step in the transepithelial transport of Na+ across frog skin. We have utilized the fluorescence properties of amiloride to study the distribution of this transport inhibitor in the ventral skin of Rana pipiens. After a 30 s exposure of 1-100 microM amiloride to the external surface of frog skin, amiloride fluorescence was evident in the cytoplasm of all cell layers of the epidermis and alveolar gland epithelium. Changes in the conditions of incubation which alter the pharmacological activity of amiloride did not affect the intracellular distribution of amiloride or the washout profile of [14C]amiloride. The presence of amiloride fluorescence in the cytoplasm prevented our examination of changes in the amiloride fluorescence at the cell surface with various conditions of incubation. Four derivatives of amiloride that differed in their ability to inhibit short-circuit current were also localized intracellularly but varied in their relative distribution among the cell layers of the epidermis. Our results indicate that when incubated at concentrations from 1 to 100 microM, a large fraction of the amiloride taken up by frog skin is not directly involved with the inhibition of passive Na+ transport at the apical surface of the stratum granulosum. The mechanism of intracellular uptake of amiloride is not clear. However, the cytoplasmic localization of amiloride could explain the action of the drug on intracellular enzymes and may account for the large proportion of non-displaceable [14C]amiloride that has been observed in frog skin.

Effect of amiloride on potential difference across rectal mucosa in cystic fibrosis patients.

This study investigated whether the altered epithelial cell function in cystic fibrosis (CF) concerns channels for passive diffusion of sodium across cell membranes. For this purpose, the potential difference (PD), which arises across the colonic mucosa from electrogenic sodium transport, was compared in CF and non-CF subjects, together with the effect on this PD from bathing the mucosa with the specific Na+ channel blocker, amiloride. The resting rectal mucosal PD did not differ significantly in the two groups. Amiloride caused a dose-dependent decrease in the PD (lumen negative to skin) in all subjects. The mean amiloride effect was less in the CF population at 10(-8), 10(-7), 10(-6), 10(-5), and 10(4) M amiloride. The decrease of transmucosal PD in CF patients was significantly less when compared with controls at 10(6) M and 10(-5) M and suggestive of a similar trend at 10(-4) M.

Structure of the tight junctions of the human eccrine sweat gland.

The human eccrine sweat gland contains two anatomically and functionally discrete segments: the secretory coil, which produces an isotonic or slightly hypertonic precursor fluid, and the coiled duct, which reabsorbs Na+ and Cl- to yield a hypotonic sweat. We examined the freeze-fracture morphology of tight junctions from isolated secretory coil and coiled duct segments to assess indirectly the contribution of paracellular ion transport in secretion and resorption in the sweat gland. In the secretory coil, tight junctions of the intercellular canaliculus and main lumen consisted of approximately 9 and 6, closely spaced, parallel or anastomosing elements, respectively. Tight junctions of the coiled duct were similar in appearance to those at the main lumen of the secretory coil. In both the secretory coil and coiled duct, and average of 2 to 3, widely spaced junctional elements were usually observed basolateral to the closely spaced junctional elements in the region corresponding to the location of the zonula adherens in Epon sections. The complexity of the tight junctions of the secretory coil exceeded what we expected for an epithelium secreting an isosmotic fluid. The elaborate tight junctions of the coiled duct support other evidence for an intermediate to high transepithelial resistance.