Downregulation of VANGL1 inhibits cellular invasion rather than cell motility in hepatocellular carcinoma cells without stimulation.

AIMS: The Wnt planar cell polarity (PCP) pathway is one of the Wnt pathways which plays a critical role in cell proliferation and fate. The VANGL1 protein is one of Wnt-PCP pathway components. It is known that Wnt-PCP pathway has major roles in cell motility but its role in hepatocellular carcinoma (HCC) progression through invasion and metastasis needs to be clarified. METHODS: We silenced VANGL1 gene expression in the HepG2 HCC cell line by stable transfection with a vector containing siRNA template for VANGL1 and investigated the change in cell invasion and motility. RESULTS: Transfected cells with the siRNA template showed significantly suppressed invasive capacity when compared to controls although cellular motility was only slightly affected. CONCLUSION: Our study showed a basal role for VANGL1 with respect to the invasive capacity of HCC cells. This suggests that the Wnt-PCP pathway may play a role in progression of HCC through cellular invasion but further studies are needed to clarify its role in cell motility.

New paraneoplastic syndrome in chronic basophilic leukemia.

Chronic basophilic leukemia (CBL) is an extremely rare disorder. A female patient presented with recurrent attacks of chills, fever and abdominal pain was found to have simultaneous cyclic oscillation in leukocyte counts and C-reactive protein values. She was initially diagnosed with familial Mediterranean fever and treated with colchicine. Diagnosis of CBL was established by morphologic studies of peripheral blood and bone marrow. Her febrile attacks recurred with marked elevation in serum interleukin-6 (IL-6) level when basophil counts climbed to peak levels during cyclic oscillation. Molecular studies by real-time PCR showed IL-6 gene expression in neoplastic basophils separated by magnetic-activated cell sorting infiltrating the bone marrow, suggesting that IL-6 is released by neoplastic basophils of an underlying CBL, resulting in a new paraneoplastic syndrome that mimics autoinflammatory disorders.

An In-House Method for Molecular Monitoring of BCR-ABL.

OBJECTIVE: At present, there are a limited number of facilities in Turkey that can provide reliable real-time quantitative(RQ)-PCR BCR-ABL results. The present study aimed to test a cost-effective, in-house method of BCR-ABL quantification,including verification of the method by RQ-PCR validation tests. MATERIAL AND METHODS: BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors, from whichcalibrators were prepared and used as templates in RQ-PCR reactions to generate standard curves. Dilutions of K562cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed. RESULTS: Standard curves were generated from calibrators. These curves were then used to calculate the BCR-ABL andABL copy numbers; in which linear BCR-ABL results were obtained. Repetitive experiments showed that our methodologywas able to detect 1 BCR-ABL positive cell from amnong 1x105 cells. CONCLUSION: The method described herein is suitable for implementation with any RQ-PCR instrument and/or kit forquantify BCR-ABL transcripts. CONFLICT OF INTEREST: None declared.

Novel functions for TAF7, a regulator of TAF1-independent transcription.

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30-40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

Expression profile of WNT, FZD and sFRP genes in human hematopoietic cells.

Identifying gene expression differences in the Wnt signaling pathway specific to leukemic cells can be hampered by the lack of verified knowledge on the expression of WNT genes in normal blood cells. In this study we aimed to determine the expression profile of human WNT, FZD and sFRP genes in normal adult bone marrow, T and B cells; along with the hematopoietic cell lines K562, HL60, Jurkat and Namalwa. Bone marrow and peripheral blood from 16 donors were evaluated and our results were compared with the GeneNote database expression arrays. In bone marrow, only WNT3, WNT10A, FZD3, FZD7 and sFRP1 genes are constitutively expressed. Lymphocytes express WNT7A, WNT9B, FZD6 and FZD7 in addition to the genes above, but T cells differ in that they lose sFRP1 expression and gain constitutive expression of WNT16. An established "normal" profile of the Wnt genes in various blood cells will provide a fundamental basis for research investigating hematopoiesis and cellular processes during leukemic transformation.

In vivo expression of MHC class I genes depends on the presence of a downstream barrier element.

Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

sFRP1 promoter methylation is associated with persistent Philadelphia chromosome in chronic myeloid leukemia.

Epigenetic silencing of sFRP genes has been shown to lead to constitutive activation of the canonical Wnt-signaling pathway. The first description of deregulated Wnt-signaling activation in a hematological malignancy was reported in chronic myeloid leukemia (CML). To investigate whether epigenetic silencing of sFRP is responsible for the observed Wnt activation in CML, we studied the methylation and mutational status of the sFRP1 promoter in 48 chronic phase CML patients. Of the 48 CML patients 41 were shown to be unmethylated, 6 patients hemi-methylated and 1 patient fully methylated at the sFRP1 promoter. Albeit observed infrequently in chronic phase CML, sFRP1 promoter methylation correlated with primary cytogenetic resistance to imatinib mesylate. sFRP1 promoter methylation may indicate a genetically more unstable form of disease resistant to therapy and provide a key biological difference in therapy resistant patients, in addition to a possible mechanism for the observed activation of canonical Wnt signaling in CML.

Beta-catenin mutations are not observed in chronic myeloid leukemia.

AIMS AND BACKGROUND: Studies reporting activated Wnt signaling in all stages of chronic myeloid leukemia (CML) have demonstrated that deregulation of the pathway plays a role in the pathogenesis of this disease. Several reports have suggested mechanisms for the deregulated Wnt signaling and beta-catenin stabilization observed in CML. One possible mechanism for beta-catenin stabilization could be the acquisition of mutations at its N-terminal domain, especially in the third exon where it is marked via phosphorylation for degradation. We sought to determine whether mutations in the third exon of the beta-catenin gene are responsible for the observed Wnt activation in CML. MATERIAL AND METHODS: We screened bone marrow specimens from 33 patients with CML in the chronic phase and also examined the K562 cell line for beta-catenin mutations. RESULTS: None of the patients nor the K562 cell line were found to carry mutations. CONCLUSION: Beta-catenin amino-terminal mutations are not observed or very rare and therefore are not the underlying mechanism of activated Wnt signaling in CML.

Isolated myelosarcoma development in an adolescent chronic myeloid leukemia patient with t(9;22)(q34;q11.2), +8, +14, +21, and der(1)(p36).

Additional chromosomal abnormalities are found in 5-20% of patients during chronic phase of chronic myeloid leukemia and in 60-80% preceding or accompanying blast crisis. These abnormalities are important in disease progression and, because they may occur before hematological and clinical symptoms, can be taken as a prognostic indicator. An adolescent with chronic myeloid leukemia initially presented with extreme thrombocytosis, increased megakaryopoiesis with dysmorphic features, and focal myelofibrosis in bone marrow examinations and then developed isolated myelosarcoma 1 year after onset, with t(9;22)(q34;q11.2), +8, +14, +21, and der(1)(p36).

Induction of apoptosis increases expression of non-canonical WNT genes in myeloid leukemia cell lines.

With the aim of determining the differential expression of WNT and FZD genes, before and after induction of apoptosis in BCR-ABL positive cells, we treated the myeloid cell line K562 and control cell line HL60 with imatinib mesylate and etoposide, and analyzed relative mRNA expression levels of WNT, FZD and sFRP genes under normal and apoptotic conditions by real-time RT-PCR. We observed marked increase in mRNA levels of FZD4, FZD5, FZD7 and WNT5b, correlating with apoptotic activity and independent of the agent or cell line used. Our results suggest the involvement of non-canonical Wnt signaling in executing programmed cell death in myeloid cell lines.

Rapid transformation of atypical myeloproliferative disorder with consistent t(8;13) to B-cell acute lymphoblastic leukemia: a case report.

8p11 myeloproliferative syndrome (EMS; also known as the stem cell leukemia syndrome-SCLL) is a rare atypical myeloproliferative disorder associated with chromosomal abnormalities involving the 8p11 chromosomal band. Translocations associated with this syndrome result in the fusion of the fibroblast growth factor receptor 1 (FGFR 1) gene with various partners, resulting in ligand independent FGFR activity. The most commonly observed translocation of this syndrome is t(8;13), which results in the expression of a chimeric ZNF198-FGFR1 tyrosine kinase. Disease phenotype associated with this translocation has some typical features such as poor prognosis, and transformation to mainly acute leukemia and non-Hodgkin lymphoma; commonly with a T-cell phenotype in which obtaining and maintenance of remission is difficult by conventional chemotherapy. We hereby present a case diagnosed as atypical chronic myeloproliferative disease with consistent t(8;13)(p12;q12) and transformed rapidly to pre-B-cell acute lymphoblastic leukemia which is a rare clinical presentation.