Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy.

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy.

Establishment and characterization of 4 new human pancreatic cancer cell lines: evidences of different tumor phenotypes.

OBJECTIVES: Pancreatic cancer still remains a challenge for its biological complexity and lack of effective therapeutic strategies. Establishing new pancreatic cancer cell lines is therefore of paramount importance to clarify its biology. METHODS: We established and characterized 4 new pancreatic cancer cell lines (PP78, PP109, PP117, and PP161) according to their genetic (K-Ras, TP53, CDKN2A, and MADH4; DNA fingerprinting; karyotype), cytostructural (cytokeratins 7, 8, 18, and 19 vimentin, and ezrin), and functional profiles (doubling time; migration assay). RESULTS: K-Ras, TP53, and CDKN2A gene alterations were detected in all 4 of them. Each cell line had a unique DNA profile revealed by DNA fingerprinting. A complex karyotype with numerous structural and numeric chromosomal abnormalities was present in each cell line. All 4 cell lines showed positivity for cytokeratins 7, 8, and 18. All but PP78 expressed cytokeratin 19, whereas vimentin was expressed only in PP117 and PP78 cells. A different ezrin cellular distribution was noticed in PP78 and PP117, being mostly located at membrane ruffles. This peculiar distribution was associated with the strongest migratory capability. CONCLUSIONS: Our results seem to confirm the pancreatic ductal adenocarcinoma heterogeneity; in fact, the same genetic abnormalities (K-Ras, TP53, and CDKN2A) may have different effects on tumor biology depending on cellular differentiation.

Apoptosis is reduced in the colonic mucosa of patients with acromegaly.

BACKGROUND: Patients with acromegaly have an increased risk of developing colonic tumours; reduced apoptosis is considered a leading mechanism in tumorigenesis. GH and IGF-1 decrease apoptosis in several cell lines including human colonic adenocarcinoma, but it is unknown whether epithelial cells of colonic mucosa of patients with acromegaly have reduced apoptosis. AIM: The aim of the study was to evaluate the degree of apoptosis in a cross-sectional study, in biopsy samples of colonic mucosa obtained from patients with acromegaly. PATIENTS AND METHODS: Eleven patients with active, untreated acromegaly (AcroUntr), 16 patients with acromegaly in remission (AcroRem) and 23 controls were enrolled in the study. Samples of colonic mucosa were obtained during colonoscopy; apoptosis was evaluated by either DNA fragmentation or terminal deoxynucleotidyl transferase assay. RESULTS: Apoptotic cells were 60.0 +/- 2.5% in samples of colonic mucosa of controls, 62.0 +/- 3.4% in those from patients with AcroRem (P = ns vs. controls), and 39.0 +/- 4.1% in those from patients with AcroUntr (P < 0.0001 vs. the other groups). Apoptosis was inversely related to serum IGF-I (r = 0.771, P < 0.001) or GH (r = 0.404, P = 0.05) levels and less to the estimated duration of disease (r = 0.384, P = 0.07). PPARgamma is considered to be a tumour suppressor gene the expression of which might be involved in colonic tumorigenesis. The expression of PPARgamma was lower in the colonic mucosa of patients with AcroUntr (2845 +/- 947 transcripts) than in that of controls (35 200 +/- 2450 transcripts) or AcroRem (29 547 +/- 3650 transcripts) (P < 0.005). The recovery of PPARgamma expression was associated with apoptosis in most cells. The lower degree of apoptosis in patients with AcroUntr was associated with a reduced expression of the antiapoptotic Bax protein. CONCLUSION: In conclusion, patients with AcroUntr have reduced apoptosis in colonic mucosa that is apparently reversed after acromegaly is cured. It is conceivable that reduced apoptosis may represent an early event in colonic tumorigenesis of patients with acromegaly.

Endothelial progenitor cells induction by short-term stimulation with phytohaemagglutinin of mononuclear cells.

BACKGROUND: The identification of circulating endothelial progenitors cells (EPCs) in the adult has forced to reconsider how new blood vessels grow in physiological and pathological conditions. Neovascularization during adult life has long been attributed to angiogenesis only. However, recent studies have revealed that peripheral blood EPCs may be recruited and incorporated into sites of active neovascularization. PURPOSE: To verify that EPCs are induced from peripheral blood mononuclear cells (PBMCs) and bone marrow derived mononuclear cells (BMMCs) upon short-term stimulation with phytohaemoagglutinin (PHA), a potent T-cell mitogen. METHODS: PBMCs and BMMCs were isolated from healthy donors. Freshly isolated or depleted of adherent cells (one day and three days of adherence) mononuclear cells (MCs) were cultured in RPMI, 10% FBS, containing PHA (10 microl/10(6) cells) for 24 h. After stimulation with PHA, clusters of adherent cells were further propagated in M199 containing L-glutammine, Hepes, 20% FBS, heparin, antibiotics and bovine retina extract for 1 and 2 weeks. PBMCs and BMMCs cultured without PHA stimulation served as controls. FACS of EPCs was performed on attached cells after 7 and 14 days of culture. RESULTS AND CONCLUSION: After stimulation of MCs with PHA for 24 h, many cells clusters were observed and around these clusters some adherent EC-like cells were observed. These cells were ovoid but a very little of these were elongated in morphology, however their number and size gradually increased during culture. However a longer time was needed for obtaining EPCs from MCs harvested after adherence. Thus this indicates that short-term signals provided by PHA must be sufficient for MCs to express the ligands necessary for the induction of EPCs but signals from monocytes/macrophages are important for a more rapid differentiation.

Different growth conditions for peripheral blood endothelial progenitors.

PURPOSE: To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs). METHODS AND MATERIALS: PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis. RESULTS: With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks. CONCLUSIONS: PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).