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During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP-like cell line HEL western blotting, RT-qPCR, lentivirus-mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation-specific (ETS) transcription factor friend leukemia integration factor 1 (Fli-1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1-mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation-specific-related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA-mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention. [ABSTRACT FROM AUTHOR]

Abstract Background The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11. Results Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis. Conclusions Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint.

Abstract Background Acute erythro- and megakaryoblastic leukaemias are associated with very poor prognoses and the mechanism of blastic transformation is insufficiently elucidated. The murine Graffi leukaemia retrovirus induces erythro- and megakaryoblastic leukaemias when inoculated into NFS mice and represents a good model to study these leukaemias. Methods To expand our understanding of genes specific to these leukaemias, we compared gene expression profiles, measured by microarray and RT-PCR, of all leukaemia types induced by this virus. Results The transcriptome level changes, present between the different leukaemias, led to the identification of specific cancerous signatures. We reported numerous genes that may be potential oncogenes, may have a function related to erythropoiesis or megakaryopoiesis or have a poorly elucidated physiological role. The expression pattern of these genes has been further tested by RT-PCR in different samples, in a Friend erythroleukaemic model and in human leukaemic cell lines. We also screened the megakaryoblastic leukaemias for viral integrations and identified genes targeted by these integrations and potentially implicated in the onset of the disease. Conclusions Taken as a whole, the data obtained from this global gene profiling experiment have provided a detailed characterization of Graffi virus induced erythro- and megakaryoblastic leukaemias with many genes reported specific to the transcriptome of these leukaemias for the first time.

Medicinal formulas are a part of the complex discipline of traditional Chinese medicine that has been used for centuries in China and East Asia. These formulas predominantly consist of the extracts isolated from herbal plants, animal parts and medicinal minerals. The present study aimed to investigate the impact of 150 formulas, used as non-prescription drugs in China, on the treatment of cancer. A formula was identified, C54, commonly used to treat sore throats, which exhibited marked growth inhibition in vitro, associated with cell cycle arrest and apoptosis. Cytotoxicity was, in part, due to the ability of C54 to inhibit the expression and function of the transcription factor, Fli-1, leading to marked inhibition of leukemic cell growth in tissue culture. However, when injected into a model of leukemia initiated by Fli-1 activation, C54 only exhibited a limited tumor inhibition. C54 also did not suppress xenograft growth of the breast cancer cell line, MDA-MB-231, orthopedically transplanted into the mammary fat pad of severe combined immunodeficiency (SCID) mice. Notably, splenomegaly and accumulation of inflammatory CD11b+/Gr1+ monocytes were observed in the tumors and spleens of C54-treated mice. As inflammation is known to accelerate tumor progression, this immune response may counteract the cell-autonomous effect of C54, and account for its limited tumor inhibitory effect in vivo. Combining C54 with an anti-inflammatory drug may improve the potency of C54 for treatment of certain cancers. The present study has highlighted the complexity of Chinese medicinal compounds and the need to thoroughly analyze their systemic effects at high concentrations in vivo. [ABSTRACT FROM AUTHOR]

Background: The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11. Results: Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis. Conclusions: Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint. [ABSTRACT FROM AUTHOR]