Fucosyltransferase VII improves the function of selectin ligands on cord blood hematopoietic stem cells.

Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6. A saturating amount of either FUT6 or FUT7, which generates comparable levels of expression of fucosylated epitopes on CB CD34(+) cells, was used for these experiments. In vitro, FUT7-treated CB CD34(+) cells exhibited greater binding to P- or E-selectin than that of FUT6-treated CB CD34(+) cells under static or physiological flow conditions. In vivo, FUT7 treatment, like FUT6, improved the early engraftment of CB CD34(+) cells in the bone marrow of sublethally irradiated NOD/SCID interleukin (IL)-2Rγ(null) (NSG) mice. FUT7 also exhibited marginally-yet statistically significant-increased engraftment at 4 and 6 weeks after transplantation. In addition, FUT7-treated CB CD34(+) cells exhibited increased homing to the bone marrow of irradiated NSG mice relative to sham-treated cells. These data indicate that FUT7 is effective at improving the function of selectin ligands on CB-HSPCs in vitro and enhancing early engraftment of treated CB-HSPCs in the bone marrow of recipients.

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

A neutralizing anti-fibroblast growth factor (FGF) 8 monoclonal antibody shows anti-tumor activity against FGF8b-expressing LNCaP xenografts in androgen-dependent and -independent conditions.

BACKGROUND: Fibroblast growth factor 8-isoform b (FGF8b) has been detected in human clinical sex-organ related cancers including hormone-refractory prostate cancer. There are, however, few relevant experimental models. A murine monoclonal anti-FGF8 antibody, KM1334, has been shown to neutralize FGF8b and inhibit the growth of androgen-dependent mouse mammary SC-3 cells in vitro and in vivo. In the present study, we evaluated the anti-tumor activity of KM1334 against androgen-dependent and -independent progression of FGF8b-expressing human prostate cancer xenografts. METHODS: FGF8b cDNA was transfected into androgen-dependent human prostate cancer cell line LNCaP, and its xenograft tumors were established subcutaneously in SCID mice with or without castration. KM1334 at the dose of 400 microg/head was injected twice weekly. RESULTS: FGF8b-expressing LNCaP cells secreted FGF8b, showed enhanced level of Erk1/2 phosphorylation, and showed more potent growth properties than mock-expressing cells in vitro and in vivo. KM1334 reduced these properties in vitro, inhibited tumorigenecity in vivo (T/C=0.33), and showed anti-tumor activity against established tumors (T/C=0.47) of FGF8b-expressing cells. FGF8b-expressing LNCaP tumors were androgen-dependent. However, they recurred as androgen-independent FGF8b positive tumors after castration. KM1334 also inhibited the growth of established FGF8b-expressing tumors in the androgen-independent states (T/C=0.47). CONCLUSIONS: These results indicate that humanized monoclonal antibodies, conserving the paratope of KM1334, are a promising candidate for therapy of FGF8b-expressing clinical prostate cancers. Follow-up studies using xenograft models with clinical FGF8b-expressing tumors are required to validate these early findings.

Benidipine, a dihydropyridine-Ca2+ channel blocker, increases the endothelial differentiation of endothelial progenitor cells in vitro.

Benidipine is a dihydropyridine-Ca2+ channel blocker used in the treatment of hypertension and angina pectoris. In the present study, we examined the effects of benidipine on the endothelial differentiation of circulating endothelial progenitor cells (EPCs) using an in vitro culture method. Peripheral blood derived mononuclear cells (PBMCs) containing EPCs were isolated from C57BL/6 mice, and then the cells were cultured on vitronectin/gelatin-coated slide glasses. After 7 days of culture, endothelial cells differentiated from EPCs were identified as adherent cells with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-labeled acetylated low density lipoprotein (Dil-Ac-LDL) uptake and lectin binding under a fluorescent microscope. Incubation of PBMCs for 7 days with benidipine (0.01-1 micromol/l) significantly increased the number of Dil-Ac-LDL+/fluorescein isothiocyanate-lectin (FITC-Lectin)+ cells. Wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor, selectively attenuated the effect of benidipine on the endothelial differentiation. In addition, benidipine treatment augmented the phosphorylation of Akt, indicating that the PI3K/Akt pathway contributed, at least in part, to the endothelial differentiation induced by benidipine. These results suggest that the treatment with benidipine may increase the endothelial differentiation of circulating EPCs and contribute to endothelial protection, prevention of cardiovascular disease, and/or an improvement of the prognosis after ischemic damage.

Inhibitory effect of the 4-aminotetrahydroquinoline derivatives, selective chemoattractant receptor-homologous molecule expressed on T helper 2 cell antagonists, on eosinophil migration induced by prostaglandin D2.

Prostaglandin (PG) D2, a major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is known to induce activation and chemotaxis in eosinophils, basophils, and T helper 2 (Th2) lymphocytes via a newly identified PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). CRTH2 is hypothesized to play an important role in the outcome of allergic responses. However, the absence of selective CRTH2 antagonists has prevented the elucidation of the role of CRTH2 in pathogenesis of allergic diseases. We now report compounds discovered as selective CRTH2 antagonists, (2R*,4S*)-N-(1-benzoyl-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylisobutyramide (K117) and (2R*,4S*)-N-(1-benzoyl-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylcyclopropanecarboxamide (K604). K117 and K604 have inhibitory effects on human CRTH2 with Ki values of 5.5 and 11 nM, respectively. The effect of these compounds is CRTH2-specific with no cross-reactivity against 15 other receptors and four arachidonic acid-metabolizing enzymes. K117 and K604 has no effect on the basal Ca2+ level and inhibited the Ca2+ response induced by PGD2 in 293EBNA cells expressing human CRTH2. Also, K117 and K604 inhibit PGD2-induced human eosinophil chemotaxis with IC50 values of 7.8 and 42.2 nM, respectively, but they do not inhibit the CC-chemokine receptor 3 agonist eotaxin-induced chemotaxis. These results indicate that K117 and K604 are highly potent and selective antagonists for human CRTH2. These compounds have possibilities to become useful tools to explore CRTH2 functions in allergic diseases.

Distribution of adenosine A(2A) receptor antagonist KW-6002 and its effect on gene expression in the rat brain.

A novel adenosine A(2A) receptor selective antagonist, KW-6002 [(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], possesses antiparkinsonian activities in rodent and primate models. In the present study, we investigated the distribution of [14C]KW-6002 in forebrain after oral administration at pharmacologically effective doses. Also, we monitored the effects of the compound on preproenkephalin (PPE) and preprotachykinin (PPT) gene expression in rat striatum. The highest level of radioactivity was observed in the striatum after oral administration of [14C]KW-6002; 30 min after 0.1 and 0.3 mg/kg, the density values in the striatum were 2.45 and 2.43 times higher than those in a reference region (frontal cortex), respectively. At the dose of 3 mg/kg, p.o., the ratio was only 1.58 and the compound was distributed more extensively in the brain. The distribution pattern and intensity of radioactivity were maintained even 90 min after the administration of [14C]KW-6002. Oral administration of KW-6002 (0.3 and 3 mg/kg/day) to rats for 14 days reversed the increased gene expression of PPE in striatum that had been depleted of dopamine by prior treatment with 6-hydroxydopamine (6-OHDA). On the other hand, KW-6002 did not alter the decreased gene expression of PPT in 6-OHDA-treated rats. These results are the first to show directly that orally administered KW-6002 is distributed selectively to the striatum and that it modulates the activity of striatopallidal enkephalin-containing neurons but not striatonigral substance P-containing neurons.