Abstract The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently, E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes, we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in vivo . Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. Published 2008 Wiley‐Liss, Inc.
A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.
1032 Although the high mobility group A1 ( HMGA1) is an oncogene that is widely overexpressed in aggressive human cancers, the molecular mechanisms that underlie transformation by HMGA1 have not been clearly elucidated. HMGA1 encodes the HMGA1a and HMG1b protein isoforms, which function in regulating gene expression. To better understand how HMGA1 overexpression leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Ninety-seven genes were differentially regulated by > 2-fold in fibroblasts overexpressing HMGA1a and 103 genes were differentially regulated by > 2-fold in fibroblasts overexpressing HMGA1b. Fifty-five of these genes were common to both the HMGA1a and HMG1b fibroblasts. From the list of genes up-regulated in cells overexpressing HMGA1a or HMG1b, the gene encoding the signaling molecule, signal transducer and activator of transcription or STAT3, was studied further because of its prominent role in malignancy. Here, we demonstrate that STAT3 is a critical downstream target of HMGA1. We show that HMGA1 binds to a conserved region of the STAT3 promoter in vivo by chromatin immunoprecipitation experiments. STAT3 mRNA and activated protein (phosphorylated STAT3) levels are increased in leukemia cells from HMGA1a transgenic mice. Activated STAT3 also recapitulates the transforming activity of HMGA1a in fibroblasts. Blocking STAT3 function induces apoptosis preferentially in leukemia cells from HMGA1a transgenic mice, but not in nontransgenic control cells. Moreover, blocking STAT3 function in human erythroleukemia (HEL) cells overexpressing HMGA1a also blocks colony formation in soft agar and cell motility. STAT3 mRNA is also increased in human acute lymphoid leukemia samples that overexpress HMGA1a , suggesting that this pathway is important in human lymphoid leukemia. Our results demonstrate that STAT3 is a direct HMGA1 target gene involved in HMGA1-mediated transformation and a potential therapeutic target in human lymphoid leukemia.
