p53 stabilization induces apoptosis in chronic myeloid leukemia blast crisis cells.

Philadelphia chromosome positive chronic myeloid leukemia has a progressive course starting in a benign phase and terminating in a blastic phase. In this study, we show that human homolog double minute 2 (HDM2) inhibition, with MI-219-a novel compound, and consequently p53 stabilization induce chronic myeloid leukemia (CML) blast crisis cells to undergo apoptosis regardless of the presence of the T315I mutation in the BCR-ABL kinase domain. The response to MI-219 is associated with the downregulation of c-Myc and the induction of p21(WAF1). The p53 target and pro-apoptotic proteins PUMA, Noxa and Bax are induced, whereas full length Bid protein decreases with increased activity of pro-apoptotic cleaved Bid, and decrease of Mcl-1 is observed by increased caspase activity. CD95/FAS (FAS antigen) receptor is also induced by MI-219, indicating that both intrinsic and extrinsic apoptotic responses are transcriptionally induced. In addition, p53 protein accumulates in the mitochondrial fraction of treated cells involved in transcription-independent induction of apoptosis. We conclude that HDM-2 inhibition with MI-219 effectively induces p53-dependent apoptosis in most blast crisis CML cells, with or without BCR-ABL mutation(s).

Hematopoietic expression of HOXB4 is regulated in normal and leukemic stem cells through transcriptional activation of the HOXB4 promoter by upstream stimulating factor (USF)-1 and USF-2.

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

ETK2 receptor tyrosine kinase promotes survival of factor-dependent FDC-P1 progenitor cells.

By virtue of its high expression in both developing hematopoietic tissues and many myeloid leukemia cells lines, the embryonic tyrosine kinase receptor ETK2 (also known as Tyro3, Sky, and Rse) has been postulated to play a role in early hematopoiesis. To investigate this role, we expressed murine ETK2 in the interleukin 3 (IL-3) dependent myeloid progenitor cell line FDC-P1 and examined its effect on growth factor dependence.ETK2 cDNAs encoding full-length or kinase domain-deleted receptor were retrovirally transduced into murine FDC-P1 cells. Survival, cell cycle status, and proliferative responses of ETK2 expressing clones were studied at normal and reduced growth factor concentrations. ETK2 was expressed as a functional tyrosine kinase of 110 and 150 kDa. This proto-oncogene altered the growth of FDC-P1 cells, allowing survival at reduced growth factor concentrations and delaying apoptosis after IL-3 withdrawal. ETK2-expressing clones contained a higher fraction of cells in the S/G2/M phases of the cell cycle, both after cytokine withdrawal and in the presence of IL-3. Furthermore, these cells had a modestly enhanced proliferative response to IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that ETK2 intracellular signaling may converge with that of hematopoietic growth factors. The effects of ETK2 expression on viability and proliferation were largely dependent on a functional intracellular tyrosine kinase domain. These results support a role for ETK2 in the survival and/or expansion of primitive hematopoietic cells and suggest that this tyrosine kinase may be implicated in myeloid leukemogenesis as well.

Identification of alternative exons, including a novel exon, in the tyrosine kinase receptor gene Etk2/tyro3 that explain differences in 5' cDNA sequences.

Protein tyrosine kinase transmembrane receptors trigger signal transduction cascades upon ligand binding, resulting in cellular proliferation, differentiation, differentiation inhibition or apoptosis depending upon the cell target. The ETK2/TYRO3 receptor is a tyrosine kinase expressed in embryonic stem cells, brain and testis that has recently been cloned by several groups. Analysis of cDNA clones isolated from several tissues shows 2 isoforms of the Etk2/tyro3 gene product that result from usage of alternative exons near the 5' end of the gene. In addition, our data suggest that a third alternative exon is positioned between these two alternative exons. This novel exon encodes yet another isoform that predicts a unique amino-terminal protein sequence. The alternative exons (exons 2A, 2B and 2C), predict three isoforms with different initiation codons, signal sequences and lengths. The existence of these multiple isoforms may be important for protein processing, translocation, or function.

Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)