MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Helicobacter pylori infection and adenocarcinoma arising in Barrett's esophagus.

Helicobacter pylori infection has been implicated in the development of chronic active gastritis and gastric neoplasms (ie, mucosa-associated lymphoid tumors and adenocarcinoma). The potential association between esophageal H pylori infection with Barrett's esophagus-associated adenocarcinoma has not been previously studied. Nineteen cases of adenocarcinoma arising in Barrett's esophagus were examined for the presence of H pylori. Barrett's esophagus was defined by the presence of metaplastic specialized-type epithelium (gastric-type epithelium with goblet cell metaplasia) in the distal esophagus. To detect the presence of H pylori, 5-microm sections, from several tissue blocks in each case, were stained with routine hematoxylin-eosin, modified Giemsa, and an antibody directed against H pylori (Dako a/s, Denmark, Lot # 111061). Stained sections were examined independently by two pathologists. All three staining methods failed to show H pylori in any of the cases examined. Sections of Barrett's esophagus (with and without dysplasia), adenocarcinoma, and stomach (when available) were uniformly negative for the presence of H pylori. We conclude that neither gastric nor esophageal infection with H pylori is a requisite for the development of adenocarcinoma in Barrett's esophagus. Moreover, it is unlikely that a significant association between H pylori infection and Barrett's-associated adenocarcinoma exists.

Collagenous gastritis associated with lymphocytic gastritis and celiac disease.

Collagenous gastritis is a rare disorder, with only 8 cases reported in the literature, 2 in children and 6 in adults. We report an additional case of collagenous gastritis in a 42-year-old man with celiac disease. A thickened (>10 microm) subepithelial collagen band with entrapped capillaries, fibroblasts, and inflammatory cells was seen in the stomach, associated with lymphocytic gastritis. The duodenal mucosa showed severe villous atrophy but no subepithelial collagen deposition. No evidence of lymphocytic or collagenous colitis was found in the colon. The patient became symptom-free on a gluten exclusion diet and showed partial improvement of histopathologic findings after 3 months. Collagenous gastritis is a rare disease, but a wider recognition of its histopathologic features and clinical associations may bring more cases to light and provide additional clues in determining its etiology and pathogenesis.

Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway.

MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients.

Tumor suppressor WWOX binds to ΔNp63α and sensitizes cancer cells to chemotherapy.

The WWOX tumor suppressor is a WW domain-containing protein. Its function in the cell has been shown to be mediated, in part, by interacting with its partners through its first WW (WW1) domain. Here, we demonstrated that WWOX via WW1 domain interacts with p53 homolog, ΔNp63α. This protein-protein interaction stabilizes ΔNp63α, through antagonizing function of the E3 ubiquitin ligase ITCH, inhibits nuclear translocation of ΔNp63α into the nucleus and suppresses ΔNp63α transactivation function. Additionally, we found that this functional crosstalk reverses cancer cells resistance to cisplatin, mediated by ΔNp63α, and consequently renders these cells more sensitive to undergo apoptosis. These findings suggest a functional crosstalk between WWOX and ΔNp63α in tumorigenesis.

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli.

The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A-beta-lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5 less than or equal to pH less than or equal to 6.0) and a low temperatures. These conditions were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn2+ ions.

Prostaglandin production and contractile response of umbilical arteries in preeclamptic pregnancies with and without intrauterine growth retardation.

Biochemical and biophysical properties of umbilical arteries from normotensive and preeclamptic pregnancies were examined. The production of prostaglandins E and F, 6-keto-PGF1 alpha, and thromboxane B2 by umbilical arteries from normotensive, mildly preeclamptic, and severely preeclamptic pregnancies were measured in incubation media at baseline and after addition of arachidonic acid. The initial baseline values of 6-keto-PGF1 alpha were decreased in the severely preeclamptic patients with intrauterine growth retardation (IUGR) but not in any of the other groups. Addition of arachidonic acid resulted in a significant increase in 6-keto-PGF1 alpha production over initial baseline in all groups except in the severely preeclamptic pregnancies without IUGR. These results suggest a differential defect in the 6-keto-PGF1 alpha metabolic pathway in severely preeclamptic patients with IUGR compared with those without IUGR. The stretch response curve to serotonin was decreased in the severely preeclamptic group with IUGR compared with the control group. The contractile response to individual vasoactive agents (serotonin, prostaglandin F2, norepinephrine, angiotensin II, and arachidonic acid) showed no significant difference between the normotensive and preeclamptic groups.