Aberrant expression of SPAG6 may affect the disease phenotype and serve as a tumor biomarker in BCR/ABL1-negative myeloproliferative neoplasms.

Sperm-associated antigen 6 (SPAG6) is a newly identified cancer-testis antigen that has been revealed to contribute to the occurrence and development of various types of human cancer, such as ovarian, bladder, breast and lung cancer. However, to the best of our knowledge, the expression levels of SPAG6 in breakpoint cluster region (BCR)/ABL1-negative myeloproliferative neoplasms (MPNs) have not been investigated previously. Using reverse transcription-quantitative PCR and different tissue staining techniques, the present study revealed that SPAG6 was expressed by MPN cells, both at the mRNA and protein levels, and that nucleated erythroid precursors and megakaryocytes expressed the highest levels of SPAG6. In addition, SPAG6, which is known as a microtubule-associated protein, was found to exhibit nucleic, cytoplasmic or both cytoplasmic and nucleic subcellular localization patterns within the same patient or cell type; however, it did not always co-localize with ß-tubulin. Furthermore, SPAG6 expression was revealed to be associated with fewer splenomegaly [P=0.015 for polycythemia vera (PV) and essential thrombocythemia (ET); and P=0.012 for primary myelofibrosis (PMF)] and myelofibrosis events (P=0.014 for PV and ET; and P=0.004 for PMF). In patients with PMF, upregulated expression levels of SPAG6 were also found to be associated with lower white blood cell counts (P=0.042) and lactate dehydrogenase levels (P=0.012), and higher hemoglobin levels (P=0.031) and platelet counts (P=0.025). In addition, the receiver operating characteristic curve analysis indicated that SPAG6 may be a potential biomarker for distinguishing MPN cases from healthy individuals. In conclusion, to the best of our knowledge, the present study is the first to report that aberrant SPAG6 expression may affect the disease phenotype and serve as a tumor biomarker in BCR/ABL1-negative MPNs.

Acquired pure red cell aplasia due to treatment with clopidogrel: first case report.

A 62-year-old woman with hypertension, type 2 diabetes mellitus, hyperlipemia and coronary heart disease started taking clopidogrel, with no addition of any other new drugs. However, with the addition of the drug, the patient was diagnosed as having acquired pure red cell aplasia (PRCA), and no any other inducing factors were detected from the patient. Furthermore, with the withdrawal of clopidogrel from the treatment, the patient recovered from the PRCA and did not recur. Therefore, we report PRCA as a rare side effect of clopidogrel for the first time.

WNT5A expression is regulated by the status of its promoter methylation in leukaemia and can inhibit leukemic cell malignant proliferation.

Although down-regulation of WNT5A expression has been reported in some types of leukaemias, the level of WNT5A expression has not been assessed in leukaemia complete remission (CR) cases, the relationship among WNT5A expression level, the status of its promoter methylation, and the curative effect of leukaemia has not been reported, and the effect of WNT5A on cell proliferation has not been assessed. In this study, we analyzed WNT5A expression in various kinds of leukaemia cases, leukaemia CR cases, non-malignant hematopoietic (NMH) cases, as well as in leukemic cell lines and CD34+ cells. The methylation status of the WNT5A promoter and the levels of the Wnt5a protein were also studied. We also investigated the effect of Wnt5a on leukemic cell proliferation. WNT5A expression level was higher in NMH but lower in leukaemia cases compared to that in CR-cases (P<0.01), and was expressed at low level in leukemic cell lines K562, U937 and Jurkat. Wnt5a protein was positive in NMH, CR cases and CD34+, but negative in leukaemia cases. WNT5A promoter was methylated in leukaemia cases and all leukemic cell lines, but not in NMH and CR cases. WNT5A expression was up-regulated after exposure to the demethylating agent 5-Aza-2'-deoxycytidine (Aza) in the K562, U937, Jurkat leukemic cell lines and in 83.3% (10/12) of CR patients after cure, respectively. The increased Wnt5a protein can inhibit K562 malignant proliferation and arrest cell cycle at the G2/M phase after exposure to Aza. These results indicate that WNT5A expression was restored in complete remission cases due to demethylation, and Wnt5a can inhibit leukaemic cell proliferation. We propose that WNT5A can act as a suppressor factor in leukemogenesis and can be used as a potential marker for curative effect assessment in leukaemia.

The Wnt5a/Ror2 noncanonical signaling pathway inhibits canonical Wnt signaling in K562 cells.

Wnt5a has been shown to be involved in cancer progression in a variety of tumor types, and regulates multiple intracellular signaling cascades; it is a representative ligand that activates a noncanonical Wnt signaling pathway. The mechanism governing how Wnt5a determines the specificity of these pathways and the relationship with tumorigenesis is still unknown. In this study, we aimed to clarify the tumor suppressor role of Wnt5a in leukemogenesis. In particular, we focused on Ror2 functioning as a Wnt5a receptor to mediate noncanonical Wnt signaling, which inhibits canonical Wnt signaling in K562 cells. We found that up-regulation of Wnt5a expression increased Ror2 expression in K562 cells and Wnt5a and Ror2 were co-expressed in the cytoplasm. Also, Wnt5a induced the intrnalization of Ror2. Co-immunoprecipitation experiments were performed to determine whether Ror2 binds to Wnt5a, and inhibits Wnt5a binding with Frizzled4 and LRP5 in Wnt5a treated K562 cells. Wnt5a had no effect on total ß-catenin expression levels, but regulated tyrosine phosphorylation of ß-catenin and translocation of ß-catenin from the cytoplasm to the nucleus. Furthermore, expression of Wnt5a was associated with suppression of ß-catenin/TCF-dependent transcriptional activity and down-regulated the expression of cyclin D1, a downstream target gene of the canonical Wnt signaling pathway. We hypothesize that Wnt5a plays the role of a tumor suppressor in leukemogenesis through the Wnt5a/Ror2 noncanonical signaling pathway that inhibits Wnt canonical signaling.

[Expression of wnt5a gene in hematologic diseases and leukemic cell lines].

This study was aimed to investigate the expression level of Wnt5a gene in some hematologic diseases and leukemic cell lines so as to provide a basis for further research of Wnt5a role and its mechanism in hematologic malignancies. The mononuclear cells of peripheral blood and bone marrow were isolated by human lymphocytic isolation solution. The expression of Wnt5a gene in specimen of 31 cases and three leukemic cell lines (Jurkat, K562, HL-60) were detected by RT-PCR. The results showed that in four out of five AML cases, negative or weak positive expressions were observed and negative expressions were observed also in K562 and HL-60 cells. Only in one AML case with complete remission and Jurkat cells the strong positive expressions were observed. The negative expression was observed in all six CML cases. In three out of four ALL cases, the expression was positive or weak positive and one negative. The expressions in two CLL cases were negative. Out of two MM cases, the expression in one was weak positive and in other was negative. Out of three lymphoma cases, the expression in one case was weak positive and in other two cases were negative. There were positive or weak positive expressions in two cases of AA, two cases of IDA, three cases of ITP, one cases of PV and ET cases. It is concluded that there have obvious down-regulated or lost expression of Wnt5a gene in 31 cases of hematologic disease and myelocytic leukemic cell lines except ALL samples. Nevertheless there have general positive expression of Wnt5a in cases of non-malignant hematologic diseases. These results suggest that the genesis of myelocytic leukemia is related to the down-regulated expression of Wnt5a.

[Differentiation phenotypes of k562 cells induced by exogenous wnt5a].

This study was aimed to investigate the effect of exogenous Wnt5a on directional differentiation of K562 cells. Wnt5a and GFP condition mediums were prepared by recombinant adenoviral vector AdWnt5a and AdGFP transfecting CHO cells. K562 cells were treated with Wnt5a and the GFP condition mediums for 1 - 7 days as Wnt5a treated group and control group respectively. The morphological changes of K562 cells were observed by light microscope and electron microscope; the differentiation phenotypes of K562 cells were identified by the cytochemical staining of POX, PAS, alpha-NAE and immunocytochemistry of CD13, CD14, CD68, and the effect of Wnt5a on cell cycle distribution of K562 cells was detected by flow cytometry. The results showed that the morphology and ultrastructure of K562 cells treated by Wnt5a displayed differentiation mature feature; both POX and PAS staining showed higher positive ratio in Wnt5a treated group than that in control group; the alpha-NAE staining also was positive, but positive intensity in Wnt5a treated group could be inhibited up to 70% by NaF. The expressions of monocytic differentiation antigens of CD14, CD68 in Wnt5a treated group were higher than those in control group, but the expression differences of granulocytic differentiation antigen CD13 between Wnt5a treated group and control group were not significant. The cell cycle in treated group was blocked at G2 phase as compared with control group. It is concluded that exogenous Wnt5a can induce K562 cells to differentiate towards mature and K562 cells treated with Wnt5a displays features of differentiation towards monocytic lineage.