The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3ß (Gsk3ß), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of ß-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and ß-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased ß-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

Expression of COX-2 and VEGF-C in cholangiocarcinomas at different clinical and pathological stages.

The aim of this study was to investigate the expression and clinical significance of cyclooxygenase 2 (COX-2) and vascular en-dothelial growth factor C (VEGF-C) in cholangiocarcinomas at differ-ent clinical and pathological stages. Eighty cholangiocarcinoma sam-ples of patients treated with surgery between January 2012 and January 2014 were collected. Immunohistochemistry was used to detect COX-2 and VEGF-C expression at different clinical and pathological stages. ELISA, real-time PCR, invasive chambers, and MTT assay were ap-plied in cultured cholangiocarcinoma cells treated with a COX-2 inhib-itor. Expression of COX-2 and VEGF-C correlated positively with the clinical TNM stage but did not correlate with the differentiation status. Inhibition of COX-2 activity reduced VEGF-C mRNA expression and secretion in cholangiocarcinoma cells and decreased their migration but not proliferation. Because of its ability to inhibit invasion, COX-2 could be a new target for treatment of cholangiocarcinoma.

Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis.

Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.

Cancer risk with alemtuzumab following kidney transplantation.

Alemtuzumab has been employed for induction therapy in kidney transplantation with low rates of acute rejection and excellent graft and patient survival. Antibody induction therapy has been linked to increased vulnerability to cancer. Data regarding malignancy rates with alemtuzumab are limited. We studied 1350 kidney transplant recipients (between 2001 and 2009) at the University of Pittsburgh Starzl Transplant Institute, for post-transplant de novo and recurrent malignancy, excluding non-melanoma skin cancer, among patients receiving alemtuzumab, thymoglobulin, and no induction therapies. Of the 1350 patients, 1002 (74.2%) received alemtuzumab, 205 (15.2%) received thymoglobulin, and 122 (9%) received no induction therapy. After excluding cancers occurring within 60 d post-transplantation, 43 (3.25%) malignancies were observed during a median follow-up time of 4.0 yr. The incidence of malignancy was 5.4% (1.09 per 100 patient-years [PY]) with thymoglobulin, 2.8% (0.74 per 100 PY) with alemtuzumab, and 3.3% (0.66 per 100 PY) with no induction (across all groups; p = 0.2342, thymoglobulin vs. alemtuzumab; p = 0.008). Thus, with the exception of non-melanoma skin cancer which we did not evaluate, alemtuzumab induction was not associated with increased cancer incidence post-kidney transplantation when compared to no induction therapy and was associated with lower cancer incidence when compared to thymoglobulin.

W-7 primes or inhibits the fMLP-stimulated respiratory burst in human neutrophil by concentration-dependent dual expression of the formyl peptide receptors on cell surface.

It was investigated why the fMLP-stimulated respiratory burst in human neutrophils was enhanced by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a considered calmodulin antagonist, at lower concentration but inhibited at higher concentration. Flow cytometric analysis on binding of the receptor to the fluorescence-labeled formyl peptide and the polymerization of actin in cells showed that the drug inhibited actin polymerization and promoted expression of the fMLP receptors on cell membrane at lower concentration, while promoted the actin polymerization and depressed the receptor expression at higher concentration. As intracellular Ca(2+) ([Ca(2+)](i)) is elevated, polymerization of actin decreases and the receptor expression increases. At normal physiological and two moderately high intracellular calcium levels, the dual effect of W-7 became less significant as [Ca(2+)](i) was elevated indicating that the dual effect is calcium-dependent. Under two extreme conditions that the intracellular calcium was either depleted or highly elevated, the dual effect disappeared but only an inhibitory effect on actin polymerization was observed. Colchicine and taxol study showed that disruption or stabilization of microtubules had no effect on formyl peptide receptor expression. The results suggest that W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of microfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca(2+)-dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration.

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.

[The relationship between fMLP induced neutrophil respiratory burst and the apoptosis of neutrophil].

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.

Extracellular Ca2+ regulates the respiratory burst of human neutrophils.

The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca2+-ATPase inhibitor, thapsigargin, was used to activate higher Ca2+ influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca2+ entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca2+ influx under normal physiological conditions. The higher Ca2+ influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra- and extracellular Ca2+ are required for full activation of the respiratory burst of human neutrophils, and the Ca2+ influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

[The clinical value of tumor marker CA50 and CA242 in digestive tract cancer].

OBJECTIVE: CA50 and CA242 are newly recognized serum tumor marker. This study is to show their value in the diagnosis of digestive tract tumor. METHODS: 164 patients with malignant digestive disease and 69 patients with benign disease were chosen. CA50 and CA242 level of these patients were measured by immunoradiometric assay. RESULTS: The total sensitivity of CA50 and CA242 are 70% and 63% respectively. Two tumor markers rarely elevated in the patients with benign disease (false positive rate CA50 7.3%, CA242 4.3%). High percentage of elevated CA242 level was recorded in the patients with pancreatic and biliary cancers (Sensitivity 79%). Both have low sensitivity in patients with liver carcinoma. CONCLUSIONS: CA50 and CA242 are valuable marker in the diagnosis of digestive tract tumor.

[Omeprazole, clarithromycin and amoxicillin therapy for Helicobacter pylori infection].

The aim of this study is to determine the eradication rate in Helicobacter pylori (Hp) infection using omeprazole 20 mg twice daily, clarithromycin 500 mg twice daily and amoxicillin 1,000 mg twice daily for one or two weeks, in comparison with colloidal bismuth subcitrate 240 mg twice daily, amoxicillin 1,000 mg twice daily and metronidazole 400 mg twice daily for two weeks. 141 patients with Hp associated chronic gastritis or duodenal ulcer were randomly divided into 3 groups. Group I (n = 48) treated with triple therapy of omeprazole, clarithromycin and amoxicillin for 1 week. Group II (n = 47) treated with the same drug and dosage for 2 weeks. Group III (n = 46) treated with triple therapy of colloidal bismuth subcitrate, amoxicillin and metronidazole for 2 weeks. The Hp status was determined by rapid urease test and histology of Warthin-Starry silver stain. The eradication rates of Group I, Group II and Group III were 89.6%, 95.7% and 71.7% respectively. The difference between Group I and Group III, or Group II and Group III was significant. The difference of eradication rate between Group I and Group II was statistically insignificant. The frequency of side effects in Group III was higher than that in Group I or Group II. Although 21.1% of the patients in Group I and II experienced a metallic taste, none discontinued the medication. Our results show that triple therapy with omeprazole, clarithromycin and amoxicillin is an effective and well tolerated treatment for eradication of Hp infection and may be an alternative regimen to the old standard triple therapy.

[Lactulose hydrogen breath test in small intestinal bacterial overgrowth].

Lactulose hydrogen breath test (LHBT) was evaluated in 21 patients suspected of having small intestinal bacterial overgrowth syndrome and 10 healthy volunteers as control. After dietary preparation and a 12-hour fast, subjects received 15g of lactulose mixed with 40% barium sulfate. The purpose of the barium meal was to reveal the position of lactulose in the intestines. End-expiratory samples of breath were taken at 15 minutes intervals at least for 4 hours. Breath hydrogen was measured with gas chromatography. A positive LHBT was defined as increase of hydrogen concentration in the breath more than 10 x 10(-6) above the baseline value before barium reached the sixth group of small intestine. Cultures were considered positive for bacterial overgrowth when anaerobic counts > or = 10(6) CFU/ml of aspirate. The procedure was carried out under sterile condition. Compared with the bacteriologic culture, LHBT has a sensitivity of 71.4%, specificity of 88.2% and accuracy of 80.6%. These results show that the LHBT is a simple, non-invasive and relatively reliable method for diagnosis of small intestinal bacterial overgrowth.

[Study on omeprazole 20 mg twice weekly in prevention of duodenal ulcer relapse].

The aim of this study is to propose a regimen of omeprazole 20 mg twice weekly for the prevention of duodenal ulcer (DU) relapse. 257 endoscopically verified DU patients entered this study. They were divided into four groups: (1) Group A, 68 patients stopped treatment after healing of DU during treatment of cimetidine 800 mg at night. (2) Group B, 72 patients healed DU after treatment of omeprazole 20 daily. Then anti-ulcer drug was withdrawn. (3) Group C, after healing of DU, 54 patients received a maintenance dosage of cimetidine 400 at night for up to 1 year. (4) Group D, after healing of DU, 63 patients received omeprazole 20 mg twice weekly (every Monday and Thursday) for up to 1 year. All the patients were followed in out-patients department for assessment of symptomatic response and side effect. Endoscopic examination were performed in those patients when any symptom related to the upper gastrointestinal tract appeared. The cumulative recurrence rates of Group A to Group D at 6 months were 44.1%, 23.6%, 22.2% and 4.8%, and that at 12 months were 76.5%, 36.1%, 27.8% and 6.3% respectively. The relapse rate of patients with omeprazole 20 mg twice a week was significantly lower than that of other groups at either 6 or 12 months. In patients with maintenance therapy of cimetidine, DU recurred more rapidly among smokers than nonsmokers, but there is no significant difference between smokers and nonsmokers receiving maintenance therapy of omeprazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose polymer increases jejunal calcium, magnesium, and zinc absorption in humans.

The present study investigated the effect of glucose polymer on jejunal calcium, magnesium, and zinc absorption in eight normal subjects, using the triple-lumen intestinal perfusion technique. For each subject, a 30-cm segment of jejunum was perfused for 60 min each with two different test solutions. When 4 mM glucose polymer was perfused net calcium absorption increased by fourfold (95 vs 488 mumol/30 cm/h), and net jejunal uptake of magnesium (393 mumol/30 cm/h) was observed, as compared to net magnesium secretion in the absence of glucose polymer. In addition, coadministration of glucose polymer doubled net zinc absorption (13 vs 29 mumol/30 cm/h). The rate of water absorption increased from 49 to 111 ml/30 cm/h. No further change in jejunal water and mineral absorption was observed when glucose polymer in the perfusate was increased from 4 to 8 mM. These results suggest that glucose polymer may have potential as an agent to significantly enhance mineral absorption.