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CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1alpha(OH)D2 and 1alpha(OH)D3.

Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl − clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl − clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl − clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl − clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl − clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

Medullary thyroid carcinoma (MTC) is associated with amyloid deposition in the surrounding tissues. MTC-positive tumor thyroid tissues surgically removed from patients were used in our study to extract amyloid. We tested the MTC extracts for the presence of amyloid by measuring fold enhancement of thioflavin T fluorescence. Transmission electron microscopic study and atomic force microscopy of MTC patient extracts revealed typical amyloid fibrils. Matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated full-length calcitonin as the constituent of the MTC amyloid from seven patients. Our results unequivocally demonstrated that full-length calcitonin is the sole constituent of amyloid in MTC.

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Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/ threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.Modification of signal transduction pathways within the synapse following neuronal activity is thought to be a mechanism underlying learning and memory in higher organisms. Protein phosphatase-1 (PP1), 1 a major protein serine/threonine phosphatase, controls numerous physiological processes in neuronal and non-neuronal tissues (1-3). Regulation of specific PP1 pools has been implicated in neuronal plasticity changes linked to simple forms of learning and memory. For example, PP1 bound to plasma membranes controls the serotonin-sensitive K ϩ channel that regulates neurotransmitter release from sensory neurons and short term sensitization of tail and siphon withdrawal reflexes in Aplysia californica (4). In the mammalian brain, PP1 is concentrated in dendritic spines where it dephosphorylates phosphoproteins like CaMKII (5-7) calcium channels (8), and NMDA (9 -11), and AMPA receptor channels (12-13) to modulate synaptic plasticity, characterized as long term potentiation (14) and long term depression (15). However, the mechanism by which PP1 is localized to specific neuronal compartments and the significance of PP1 localization for neuronal signaling remains largely unknown.Studies of cellular and molecular mechanisms that control PP1 activity in all eukaryotic cells have emphasized a recurrent regulatory paradigm, whereby the PP1 catalytic subunit is associated with distinct regulatory subunits that define its subcellular distribution and modify its biochemical properties. In this manner, the regulatory subunits dictate the diverse functions of PP1 in the eukaryotic cell (16). Biochemical isolation of PP1 complexes (17), use of yeast two-hybrid protein interaction screens (18), and genetic manipulations of the PP1 catalytic subunit in y...

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