Localization of plasma membrane bound NTPDases in the murine reproductive tract.

Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

Enhanced expression of alpha(1,3)-fucosyltransferase genes correlates with E-selectin-mediated adhesion and metastatic potential of human lung adenocarcinoma cells.

Alpha(1,3)- and alpha(1,4)-fucosylated oligosaccharides such as sialyl-Lewis(x) (sialyl-Le(x)) and sialyl-Lewis(a) (sialyl-Le(a)) have been reported to participate in tumor cell adhesion to activated endothelium. We examined by cytofluorometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a), and sialyl-Le(a) on the surface of two human lung adenocarcinoma cell lines with different lung colonization potential. High expression levels of all of these antigens were detected in the metastatic HAL-8Luc cells, whereas the closely related nonmetastatic HAL-24Luc cells only expressed the sialyl-Le(a) and sialyl-Le(x) dimeric antigens, both at lower level than in HAL-8Luc cells. Five alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-T) controlling the synthesis of these molecules have been identified to date (Fuc-TIII-Fuc-IVII). The expression of these five genes was also higher in the metastatic cells than in the nonmetastatic counterparts as was shown by Northern blot analysis. In vitro adhesion assays showed that only the metastatic cell line adheres significantly to E-selectin-expressing human endothelial cells. Moreover, monoclonal antibody (mAb) blockade of E-selectin completely abolished tumor cell binding. Adhesion inhibition experiments using mAbs against sialylated fucosylated oligosaccharides expressed on tumor cells indicated that these antigens are involved in the binding. Anti-sialyl-Lex(x) mAb (CSLEX-1) inhibited adhesion by 85%; it had the most pronounced inhibitory effect. These findings suggest that the overexpression of alpha(1,3)-Fuc-T genes in the metastatic HAL-8Luc cells, compared with HAL-24Luc cells, results in an enhanced surface display of fucosylated oligosaccharides, which contributes to the adhesive capacity of these cells to the activated endothelium and correlates with their lung colonization potential.

Changes in expression and activity levels of ecto-5'-nucleotidase/CD73 along the mouse female estrous cycle.

AIM: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5'-nucleotidase (CD73), the enzyme generating adenosine from AMP. METHODS: Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5'-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. RESULTS: Ecto-5'-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5'-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. CONCLUSION: The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility.

Identification of semaphorin E gene expression in metastatic human lung adenocarcinoma cells by mRNA differential display.

UNLABELLED: Human lung adenocarcinoma cell lines HAL-8Luc and HAL-24Luc differ in their metastatic potential. HAL-8Luc cells metastasize to lungs when injected either intravenously or intramuscularly. in mice while HAL-24Luc cells do not. The differential display method is used to identify genes differentially expressed between the two cell lines and the findings are extensively discussed. BACKGROUND: Lung cancer is the leading form of cancer in most countries, and metastasis is the main cause of death in oncological patients. The metastatic phenotype of tumor cells is the result of genetic events altering the RNA and protein expression of normal cells. Our objective was to identify genes expressed differentially between metastatic and nonmetastatic human lung adenocarcinoma cells that might be used as a prognostic factor. METHODS: The differential display technique was used to compare the RNA expression patterns distinguishing metastatic (HAL-8Luc) and nonmetastatic (HAL-24Luc) human lung adenocarcinoma cells, two genetically close cell lines. RESULTS: Differential expression of three cDNAs was confirmed by Northern blot analysis. Two sequences corresponding to a putative splicing factor and a proliferation-related factor cDNAs were underexpressed in the metastatic cells relative to the nonmetastatic ones. Interestingly, we found that human semaphorin E mRNA was several fold overexpressed in the metastatic cells. This recently identified gene encodes a protein whose expression has been related to several cell survival mechanisms as well as to immunosuppression. CONCLUSION: Our results point to the relevance of semaphorin E in metastatic spread of human lung adenocarcinoma cells.

Overexpression of alpha(1,3)-fucosyltransferase VII is sufficient for the acquisition of lung colonization phenotype in human lung adenocarcinoma HAL-24Luc cells.

Metastatic human lung adenocarcinoma HAL-8Luc cells display an enhanced expression of alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-Ts) compared with their non-metastatic counterpart HAL-24Luc cells. This correlates with an increased surface expression of Lewis(x) (Le(x))- and Lewis(a) (Le(a))-related molecules and an in vitro enhanced adhesive capacity to E-selectin-expressing endothelial cells (Martin-Satué et al (1998). Cancer Res 58: 1544-1550). In the present work we have stably transfected HAL-24Luc cells with the cDNAs for the alpha(1,3)-Fuc-TIV and VII enzymes and analysed by flow cytometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a) and sialyl-Le(a). Fuc-TVII transfectants exclusively overexpress sialyl-Le(x) while Fuc-TIV-transfected cells only overexpress the Le(x) oligosaccharide. We show that solely Fuc-TVII transfectants are able to adhere to interleukin-1beta-stimulated HUVEC monolayers. We also demonstrate that Fuc-TVII overexpression in HAL-24Luc cells is sufficient for the acquisition of the lung colonization phenotype. This is the first report directly showing the contribution of an alpha(1,3)-Fuc-T to the metastatic behaviour of human lung adenocarcinoma cells.

ATP crossing the cell plasma membrane generates an ionic current in xenopus oocytes.

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.