15-Lipoxygenase 2 (15-LOX2

15-Lipoxygenase 2 (15-LOX2 15-Lipoxygenase 2 (15-LOX2)1 is a recently cloned lipoxygenase that shows the highest homology (ϳ80% amino acid identity) to murine 8-LOX, with ϳ40% identity to human 5-LOX, 12-LOX, or 15-LOX1 (1). It has at least three splice variants (termed 15-LOX2sv-a/b/c) (2, 3) and metabolizes preferentially arachidonic acid (AA) to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) (1). 15-LOX2 shows an interesting tissue expression pattern, i.e. mainly in prostate, lung, skin, and cornea (1-3). This tissue-restricted expression pattern suggests that 15-LOX2 may play a role in the normal development and its abnormal expression/function may contribute to tumorigenesis in these organs. Indeed, work by indicates that 15-LOX2 mRNA, protein expression, and enzymatic activity are decreased in high grade prostate intraepithelial neoplasia (PIN) and prostate cancer (PCa), and the expression levels of 15-LOX2 are inversely correlated with the pathological grade (Gleason scores) of the patients. We recently reported that 15-LOX2 is a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells (3). These observations (3-6) together raise the possibility that 15-LOX2 may represent an endogenous prostate tumor suppressor, and its down-regulation may contribute to PCa development. Here we provide experimental data in support of this possibility as restoration of 15-LOX2 expression inhibits PCa cell proliferation in vitro and tumor development in vivo. We further show that the tumor-suppressive functions of 15-LOX2 do not necessarily depend on the AA-metabolizing activity and nuclear localization as 15-LOX2sv-b, a splice variant that does not metabolize AA and is mostly excluded from nucleus, demonstrates similar inhibitory effect on PCa development. MATERIALS AND METHODSCells and Reagents-Six primary NHP cell strains, NHP1-NHP6, were prepared from six different donors. NHP1, NHP3, NHP4, and NHP6 cells were obtained from Clonetics (Walkersville, MD), and NHP2 and NHP5 cells were generated as previously described (7-9). These cells were cultured in serum-free, PrEBM medium (Clonetics)

Recent advances in the biology of stem cells has resulted in signifi cant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the diffi culty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the current status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profi les of the intestinal cellular differentiation.

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Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.