Neutrophil gelatinase-associated lipocalin regulates intracellular accumulation of Rh123 in cancer cells.

Multidrug resistance (MDR) is a major problem facing patients with cancer. Although Neutrophil gelatinase-associated lipocalin (NGAL) is highly expressed in various cancers, the possible role of NGAL in MDR is still obscure. In this article, we evaluated the effect of NGAL on Rh123 accumulation in cancer cells. NGAL was first down-regulated by short hairpin RNA-mediated interference. In correlation with the reduced NGAL expression, intracellular Rh123 accumulation was significantly decreased. We finally observed that inhibiting both of the ERK1/2 and p38 MAPK could seriously down-regulate NGAL expression and also decrease the intracellular accumulation of Rh123, indicating that NGAL-mediated Rh123 accumulation is regulated by the phosphorylation of ERK1/2 and p38 MAPK. Pretreatment of MDA-MB-231 with NGAL recombinant protein and antibody had significant effects on the intracellular accumulation of Rh123, whereas little effect was observed in K562 cells treated with the same method, suggesting that NGAL was involved in the regulation of Rh123 accumulation in these two types of cancers, although different pathways. Here we provide new evidence that directly shows the possibility of small chemical substances Rh123 intracellular accumulation that is regulated by NGAL. These results suggest the possibility of NGAL involvement in drug transportation and cancer MDR formation, and indicate the potential of NGAL in cancer therapy.

Analysis of monocyte and histiocytic cell populations in bone marrow of patients with confirmed and suspected cases of reactive histocytosis.

OBJECTIVE: To describe characteristics of monocytes and histiocytes in the bone marrow of patients with a confirmed and suspected diagnosis of reactive histiocytosis. METHODS: 14 patients with a confident diagnosis of reactive histiocytosis or with a suspected diagnosis were inpatients at the Tianjin Blood Diseases Hospital between 2008 and 2012. Nucleated cells from bone marrow were observed by light microscopy - morphologically and immunohistochemically for histiocyte antigens - and ultrastructurally by transmission electron microscopy. RESULTS: Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were significantly increased in 9, 9, 5, 3, 3 and 2, respectively, of the 14 cases. Atypical histiocytes expressed some morphological characteristics of promonocytes. CONCLUSION: Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were increased in different relative degrees in patients with bone marrow reactive histiocytosis or suspected reactive histiocytosis. The increase in numbers of monocytes, atypical histiocytes and macrophages was a particularly significant feature. It is argued that atypical histiocytes with immature monocyte features might be precursors of hemophagocytes, reticular cells or dendritic cells.

Nuclear accumulation of calcineurin B homologous protein 2 (CHP2) results in enhanced proliferation of tumor cells.

The interaction between calcineurin B homologous protein 2 (CHP2) and Na(+) /H(+) exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivation-induced death. Although four putative EF-hands in CHP2 had been predicted for years, Ca²(+) -binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca²(+) -binding motifs required for the stable formation of CHP2/NHE1 complexes, we developed a mutagenesis-based assay in PS120 cells. We found that (45) Ca²(+) bond to two EF-hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca²(+) -binding affinity of CHP2. Co-immunoprecipitation and distribution of GFP-tagged CHP2-EF3m/4m also indicated that Ca²(+) affected the membrane location of CHP2 to interact with NHE1. The C-terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation.

Multiple organ invasion by viruses: pathological characteristics in three fatal cases of the 2009 pandemic influenza A/H1N1.

To further understand the pathological characteristics of multiple organ involvement of the 2009 pandemic influenza A/H1N1 infection, tissues of bronchial mucosa, lung, myocardium, gastrocnemius, and liver from 3 patients with fatal A/H1N1 infections were investigated by light microscopy and transmission electron microscopy. In all 3 patients, bronchial mucosa showed necrotizing bronchiolitis, epithelial necrosis and desquamation, and squamous metaplasia, while lung consolidation or fibrosis was identified. Myocardium and gastrocnemius exhibited focal necrosis and fibrosis, surrounded by muscle cells showing features of cell damage. In liver, there was widespread fatty degeneration and necrosis, most often around the central lobular vein and portal area. Viral particles were found in all samples, frequently located in endothelium, epithelium, and muscle cells. The observations demonstrate that in fatal cases of A/H1N1 infection, viruses not only infect the respiratory system, but also engage in multiple organ invasions, causing pathologic changes.

Myofibroblast transformation in metastatic extramedullary chronic myeloid leukemia: a case report.

Primary and metastatic carcinomas have a reactive stroma characterized by many myofibroblasts. These cells have also been documented in nonepithelial malignancies, such as sarcomas, malignant melanoma, and lymphoid tumors but in generally far fewer numbers. In non-Hodgkin's lymphoma, Hodgkin's disease, and leukemia, myofibroblasts are rather rarely documented. In particular, there appear to be no reports of myofibroblasts in either primary bone-marrow/peripheral blood leukemia or secondary deposits of leukemia. In this paper, a case of a relapsed chronic myeloid leukemia appearing in an inguinal lymph node is described, containing many myofibroblasts. The case is detailed and presented with a discussion on the role of myofibroblasts in the progression of nonepithelial cancers.

Invasion of erythroblasts by Pasmodium vivax: A new mechanism contributing to malarial anemia.

Severe malarial anemia causes considerable mortality and morbidity in endemic areas. Possible mechanisms underlying the anemia include lysis of parasitized and nonparasitized red cells as well as parasite product-mediated effects on erythropoiesis. The latter include suppression of erythropoiesis, dyserythropoiesis, and ineffective erythropoiesis. Present transmission electron microscope data in two cases of Pasmodium vivax malaria show a hitherto undescribed mechanism contributing to malarial anemia, namely, infection of erythroblasts by parasites and their subsequent degradation. No parasites were detected in the peripheral blood but parasites were found in the bone marrow. These findings emphasise the value of bone marrow examination in the diagnosis and eradication of malaria.

[Bioinformatics Analysis and Verification of Aging-Related Genes of Bone Marrow Mesenchymal Stem Cells in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To screen and verify the differentially expressed genes related with aging of bone marrow mesenchymal stem cells (BM-MSCs) in acute myeloid leukemia (AML) patients by bioinformatics, so as to provide new molecular markers for the research and clinical treatment of AML. METHODS: The gene expression profiling chip related with BM-MSCs in AML patients in our hospital and the gene chip GSE84881 selected from NCBI database GEO were used for data analysis and exploration. The DAVID analysis software was used to perform gene ontology (GO) enrichment analysis and KEGG pathway enrichment analysis. Furthermore, the differentially expressed genes related with aging of BM-MSCs in AML patients were identified. Bone marrow samples were collected and MSCs were amplified in vitro, and RT-PCR was used to verify the differentially expressed genes, which should be further identified with senescence-associated ß-galactosidase staining and MTT cell proliferation assays. RESULTS: A total of 247 differentially expressed genes were screened out by bioinformatics methods, including genes of 132 up-regulated expression and 115 down-regulated expression. Six differentially expressed genes related with aging of BM-MSCs in AML patients were screened out, including the genes of up-regulated expression, COL3A1 (P<0.05), CRYAB (P<0.01), DCN (P<0.05), and the genes of down-regulated expression, including CCL2 (P<0.05), CTSC (P<0.01) and IL6 (P<0.05). These 6 differentially expressed genes were consistent with data from chip assays, and which was significantly correlated with aging of BM-MSCs in AML patients. Meanwhile, the positive rate of senescence-associated ß-galactosidase staining in BM-MSCs of AML patients was significantly different from that of healthy donors (P<0.01). MTT cell proliferation assay showed that BM-MSCs in AML patients had proliferative ability lower than the healthy donors' BM-MSCs. CONCLUSION: The data here suggest novel clues for the clinical research and treatment of BM-MSCs aging in AML patients.

Ultrastructural characteristics of bone marrow in patients with hematological disease: a study of 13 cases.

There are few transmission electron microscopic studies on bone marrow biopsies of patients with hematological disease owing to the difficulty of overcoming the artifacts of decalcification. Following the fixation of bone marrow biopsies thoroughly before a mild decalcification procedure, ultrastructural studies were performed on 13 patients with varied hematological diseases. Notable features included blood cell disorganization, fibroblast activation, myofibroblast transformation, as well as accumulation of collagen and extracellular amorphous matrix. In addition, excessive blood cell death in leukemia, apoptosis, and macrophage phagocytosis in myelodysplastic syndrome and polycythemia vera, as well as degranulation of eosinophils and megakaryocytes in chronic idiopathic myelofibrosis were predominant, respectively. The observations suggest that polyclonal fibroblast proliferation and extracellular matrix accumulation may result from inflammation resulting from excessive cell death and active material release of blood cells in the bone marrow of patients with hematological disease.

Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction.

BACKGROUND: Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line. METHODS: By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions. RESULTS: A slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells. CONCLUSIONS: The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

[Effect of CIAPIN1 gene on proliferation of K562 cells].

The study was aimed to investigate the effect of CIAPIN1 gene on the proliferation of chronic myeloid leukemia (CML) cell line K562. The shRNA eukaryotic expression vector targeting CIAPIN1 gene was constructed and transfected into K562 cells. The inhibitory efficiency on K562 cells was detected by real-time PCR and Western blot; the proliferative activity of K562 cells was detected by MTT assay; the number and size of colonies were assessed by using colony-forming test; the tumorigenic potential was tested in vivo by using nude mice. The results indicated that as compared with control group, the CIAPIN1 gene expression statistically decreased; the proliferative activity of K562 cells in interference group was distinctly weakened; the number and size of colonies were significantly reduced; the tumorigenic potential was also lowered in vivo. It is concluded that inhibition of CIAPIN1 expression can inhibit K562 cell proliferation in vitro and in vivo.

[Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

[Expression of RHBDD1 gene in patients with chronic myeloid leukemia and its clinical significance].

This study was aimed to investigate the expression of RHBDD1 gene in patients with chronic myeloid leukemia (CML) and explore its clinical significance. The relative expression levels of RHBDD1 in bone marrow mononuclear cells of healthy controls and CML patients were detected by using real time PCR. The results showed that the expression level of RHBDD1 in CML patients was significantly higher than that in healthy controls. The expression level of RHBDD1 in CML patients with negative BCR/ABL p210 was remarkably higher than that in patients with positive BCR/ABL p210. In patients ≥ 50 years old RHBDD1 expression was lower than the patients < 50 years old. There were no significant relation of RHBDD1 expression with sex of patients. It is concluded that RHBDD1 gene may be involved in the pathogenesis and progression of CML, particularly reflects in the pathogenesis of the patients with negative BCR/ABL p210.

[Inhibition of NHE1 down-regulates IL-8 expression and enhances p38 phosphorylation].

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

[Dexamethasone impairs immunosuppressive ability of mesenchymal stem cells].

This study was aimed to investigate the effect of dexamethasone (Dex) on immunosuppressive ability of mesenchymal stem cells (MSC) during expansion and differentiation of MSC. MSC were cultured in 96-well flat-bottom plates. Proliferation assays were performed by using the BrdU colorimetric ELISA Kit. To explore the effect of Dex on MSC immunosuppressive ability, MSC were firstly cultured in complete culture medium for 14 d with Dex (10 nmol/L), and then, peripheral blood mononuclear cells (PBMNC) were co-cultured with MSC in 96-well flat-bottom plates for 3 d. Phytohemagglutinin A (PHA, 10 µg/ml) was used to stimulate activation of PBMNC. The concentrations of IFN-γ in culture supernatants was detected by ELISA. The results indicated that there was no obvious difference in representative phenotypes of MSC between experimental and control groups after MSC were treated with low concentration of Dex (10 nmol/L) for 14 d, but the suppression of Dex-treated MSC on lymphocyte activation in same concentration of cells was significantly reduced as compared with control group. After the Dex-treated MSC were co-cultured with IFN-γ for 12 h, the immunoregulatory ability of MSC was recovered in a certain degree. It is concluded that the Dex impairs the immunosuppressive ability of MSC, the IFN-γ can protect and reverse the immunosuppressive ability of MSC impaired by Dex, so that, when the immunoregulatory activity of MSC is investigated, it is necessary to avoid adding Dex in the culture medium.

[Increasing sensitivity of leukemia cells to imatinib by inhibiting NHE1 and p38MAPK signaling pathway].

This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.

[Expression of CIAPIN1 gene in BMMNC of patients with leukemia].

This study was aimed to investigate the expression level of CIAPIN1 mRNA in leukemia patients and explore its significance in leukemias. The fresh bone marrow was collected from 112 newly diagnosed leukemia patients, the total RNA was extracted by means of TRIzoL, the cDNA was synthesized, the expression of CIAPIN1 mRNA was detected by real-time quantitative PCR using ß-actin as internal reference; 10 normal healthy persons were selected as controls. The results showed that the expression of CIAPIN1 mRNA was statistically higher in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and chronic phase chronic myeloid leukemia (CML) patients than that in normal persons (p < 0.05); but there was no statistical difference between chronic lymphocytic leukemia (CLL) and normal persons (p > 0.05). It is concluded that the CIAPIN1 gene higher expresses in MNC of newly diagnosed leukemia patients, up-regulation of CIAPIN1 expression may play an important role in pathogenesis of leukemia.

[Early apoptosis of HL-60 cells induced by anti-CD44 McAb A3D8 inhibiting ERK1/2-upregulated Bim expression].

This study was aimed to investigate the effects of anti-CD44 mAb A3D8 on proliferation and apoptosis of AML cells, to explore the mechanism of ERK1/2 and Bim in this process. Effect of the anti-CD44 mAb A3D8 on the HL-60 cell proliferation was assayed with MTT method, the change of mitochondrial transmembrane potential of HL-60 cells was analyzed by flow cytometry. The mRNA expression of Bim was determined by real-time quantitative RT-PCR. Western blot was used to detect the protein expression of p-ERK1/2. The results showed that mAb A3D8 could remarkably inhibit the proliferation capacity of the HL-60 cells in a dosage- and time-dependent ways. The mitochondrial transmembrane potential in HL-60 cells treated with A3D8 (3.0 µg/ml) was significantly decreased as compared with the control cells. Furthermore, the mRNA expression of Bim was much higher than that in controls. Expression of the p-ERK was much lower than that of the controls. It is concluded that anti-CD44 mAb A3D8 can inhibit the proliferation and induce the apoptosis of HL-60 cells, mechanism of which is enhancing the expression of Bim via inhibiting p-ERK1/2.

[Inhibition of NHE1 promotes hypoxia-induced differentiation of K562 leukemic cells].

This study was purposed to investigate the effect of hypoxia microenvironment on K562 leukemic cell differentiation, and characteristics of NHE1 involvement in this process. The K562 cells were treated with hypoxia-mimical agent CoCl2 or under actual hypoxia culture, and the specific NHE1 inhibitor Cariporide was used to inhibit NHE1 activity. The fluorescent probe BCECF was used for pH(i) measurements. Gene expression was analyzed by RT-PCR. The morphological characteristics was determined by Wright's staining. Signaling pathways were detected by Western blot using phosphospecific antibodies. The results indicated that the hypoxia or mimetic hypoxia favored K562 cells differentiation with up-regulation of C/EBPα. Moreover, treatment with Cariporide under hypoxia synergistically enhanced leukemia cell differentiation. Treatment with Cariporide increased levels of phosphorylated ERK5 and P38 mitogen-activated protein kinase (MAPK). It is concluded that the hypoxia or mimetic hypoxia can induce the differentiation of K562 cells, the inhibition of NHE1 activity can promote the hypoxia-induced K562 cell differentiation. The enhancement of hypoxia-induced K562 differentiation by Cariporide via MAPK signal pathway suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukemias.

[Reversal of multidrug resistance in HL-60, MSC and CD34(+) cells from umbilical cord blood by sustained intracellular acidification].

The aim of this study was to investigate the effect of intracellular acidification on accumulation of rhodamine 123 (rh123) in non-mature cells with none or low expression of multidrug resistance MDR1. The expression of MDR1 mRNA was detected by real-time quantitative RT-PCR. Confocal laser microscopy was used to determine the calibration curve of intracellular acidification (pHi). MTT assay was used to detect the cytotoxicity of intracellular acidification on HL-60, MSC and CD34(+) cells from umbilical cord blood. Flow cytometry was applied to measure the influence of intracellular acidification. The results indicated that the intracellular acidification had no obvious cytotoxicity on HL-60, MSC and CD34(+) cells. The acidification resulted in the increased rhodamine 123 accumulation in HL-60, MSC and CD34(+) cells without P-gp activity. Moreover, the more primitive cells, the less accumulation of intracellular Rh123 were observed. It is concluded that the intracellular acidification can reverse the MDR of HL-60, MSC and CD34(+) cells.