Citations

Abstract Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA ≥2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 ± 1.7–fold), transforming growth factor-α (14.4 ± 5.0–fold), heparin-binding EGF-like growth factor (4.5 ± 1.4–fold), cyclin D1 (4.6 ± 0.7–fold), and cyclooxygenase-2 (COX-2; 9.3 ± 4.2–fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 ± 1.2–fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 ± 1.3–fold), phosphoactive ErbB2 (2.6 ± 0.4–fold), phosphoactive extracellular signal-regulated kinase (3.7 ± 1.1–fold), and cyclin D1 (3.4 ± 0.8–fold; P < 0.05). Ras was activated in 46% of ACF (3.2 ± 0.4–fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF. (Cancer Res 2006; 66(11): 5656-64)

Abstract Histopathologic classification plays a key role in separating multiple forms of idiopathic interstitial pneumonia into clinically meaningful categories with important differences in natural history, prognosis, and treatment. Microscopic criteria in diagnosis of these entities include the pattern and microanatomic distribution of inflammation, fibroblast proliferation, collagen deposition, and architectural remodeling. Usual interstitial pneumonia (UIP) defines idiopathic pulmonary fibrosis and is the most common of the idiopathic interstitial pneumonias. UIP has distinctive morphologic features that allow precise diagnosis in classical cases. Other forms of idiopathic interstitial pneumonia include desquamative interstitial pneumonia, respiratory bronchiolitis–associated interstitial lung disease, acute interstitial pneumonia, and nonspecific interstitial pneumonia. These latter categories differ from UIP in that the histopathologic findings do not, by themselves, allow specific diagnosis in most cases and require careful correlation with clinical and radiologic findings.

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.

Cholangiocytes line the intrahepatic bile ducts and regulate salt and water secretion during bile formation, but the mechanism(s) regulating ductal water movement remains obscure. A water-selective channel, the aquaporin CHIP, was recently described in several epithelia, so we tested the hypothesis that osmotic water movement by cholangiocytes is mediated by CHIP. Isolated rodent cholangiocytes showed a rapid increase in volume in the presence of hypotonic extracellular buffers; the ratio of osmotic to diffusional permeability coefficients was > 10. The osmotically induced increase in cholangiocyte volume was inversely proportional to buffer osmolality, independent of temperature, and reversibly blocked by HgCl2. Also, the luminal area of isolated, enclosed bile duct units increased after exposure to hypotonic buffer and was reversibly inhibited by HgCl2. RNase protection assays, anti-CHIP immunoblots, and immunocytochemistry confirmed that CHIP transcript and protein were present in isolated cholangiocytes but not in hepatocytes. These results demonstrate that (i) isolated cholangiocytes and intact, polarized bile duct units manifest rapid, mercury-sensitive increases in cell size and luminal area, respectively, in response to osmotic gradients and (ii) isolated cholangiocytes express aquaporin CHIP at both the mRNA and the protein level. The data implicate aquaporin water channels in the transcellular movement of water across cholangiocytes lining intrahepatic bile ducts and provide a plausible molecular explanation for ductal water secretion.

To investigate the molecular basis of regional variation in expression of brain acetylcholinesterase (AChE; EC 3.1.1.7), steady-state levels of AChE activity and mRNA were examined. Relative AChE activity in Triton extracts from six areas of the rat brain varied as follows: cortex < cerebellum < medulla < pons-midbrain < thalamus < striatum. In contralateral samples from the same brains, AChE mRNA was assessed by Northern blotting with random-primed 32P-labeled cDNA. The regional abundance of the major 2.4-kb AChE transcript differed from that of the enzyme activity: cortex < striatum < cerebellum < medulla < thalamus < pons-midbrain. In situ hybridization with a 33P-labeled antisense AChE oligonucleotide provided evidence for high levels of AChE message in cells of the nucleus basalis, nucleus accumbens, neostriatum, substantia nigra, motor nucleus of the facial nerve, and spinal nucleus of the trigeminal nerve. In the caudate-putamen, large, heavily labeled neurons were not numerous, but they were approximately as frequent as the cholinergic interneurons revealed by choline acetyltransferase immunocytochemistry. The relatively low number of these AChE-expressing cells probably explains the relative dearth of AChE mRNA-like material in the neostriatum.

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated cells from human giant cell tumors. These osteoclast-like cells have been examined for evidence of estrogen receptors (ERs) and responses to 17 beta-estradiol (17 beta-E2). Analysis of giant-cell RNA demonstrated expression of ER mRNA. Furthermore, immunoblot analysis revealed that the giant cells contained a 66-kDa protein that was recognized by a monoclonal antibody specific for the human ER. When isolated multinucleated cells were cultured on slices of bone, there was a dose-dependent decrease in resorption in response to treatment detectable at 10 pM 17 beta-E2. Treatment with 10 nM 17 alpha-estradiol or vehicle (control) did not inhibit resorption. Moreover, the multinucleated cells isolated from these tumors had decreased mRNA levels for cathepsin B, cathepsin D, and tartrate-resistant acid phosphatase (TRAP) as well as secreted cathepsin B and TRAP enzyme activity in response to treatment with 10 nM 17 beta-E2. In contrast to these data, no change in gene expression was detected in mononuclear cells from these tumors in response to 17 beta-E2 treatment. These data support the proposition that human osteoclasts are target cells for estrogen and that estrogen can inhibit bone resorption by human osteoclasts.

After therapeutic hormone deprivation, prostate cancer cells often develop androgen-insensitive growth through mechanisms thus far undefined. Neuropeptides have been previously implicated as growth factors in some prostate cancers. Here, we demonstrate that androgen-sensitive LNCaP human prostate cancer cells produce and secrete neurotensin following androgen withdrawal. We show that while LNCaP cells express the neurotensin receptor, only androgen-deprived cells exhibit a growth response to exogenous neurotensin. We further demonstrate that androgen-stimulated cells may be refractory to exogenous neurotensin due to androgen induction of a metalloprotease active toward neurotensin. Thus, prostate cancer cells deprived of androgen develop an alternative autocrine growth mechanism involving neurotensin.