No abstract

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiologic expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia aberrant HB9 expression is the only known molecular hallmark and assumed to be a key factor in leukemic transformation. However, up to now only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53-p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated β-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+ hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9 dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia for the first time.

AC133 is a prominent surface marker of CD34+ and CD34- hematopoietic stem/progenitor cell (HSPC) subsets. AC133+ HSPCs contain high progenitor cell activity and are capable of hematopoietic reconstitution. Furthermore, AC133 is used for prospective isolation of tumor-initiating cells in several hematological malignancies. Nucleolin is a multifunctional factor of growing and cancer cells, which is aberrantly active in certain hematological neoplasms, and serves as a candidate molecular target for cancer therapy. Nucleolin is involved in gene transcription and RNA metabolism and is prevalently expressed in HSPCs, as opposed to differentiated hematopoietic tissue. The present study dissects nucleolin-mediated activation of surface AC133 and its cognate gene CD133, via specific interaction of nucleolin with the tissue-dependent CD133 promoter P1, as a mechanism that crucially contributes to AC133 expression in CD34+ HSPCs. In mobilized peripheral blood (MPB)-derived HSPCs, nucleolin elevates colony-forming unit (CFU) frequencies and enriches granulocyte-macrophage CFUs. Furthermore, nucleolin amplifies long-term culture-initiating cells and also promotes long-term, cytokine-dependent maintenance of hematopoietic progenitor cells. Active β-catenin, active Akt and Bcl-2 levels in MPB-derived HSPCs are nucleolin-dependent, and effects of nucleolin on these cells partially rely on β-catenin activity. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis.

The epigenetic regulation by DNA methylation is an important mechanism to control the expression of stem cell factors as demonstrated in tumor cells. It was recently shown that hepatic stellate cells (HSC) express stem/progenitor cell factors and have a differentiation potential. The aim of this work was to investigate if the expression of stem cell markers is regulated by DNA methylation during activation of rat HSC. It was found that CD133, Notch1, and Notch3 are regulated via DNA methylation in HSC, whereas Nestin shows no DNA methylation in HSC and other undifferentiated cells such as embryonic stem cells and umbilical cord blood stem cells from rats. In contrast to this, DNA methylation controls Nestin expression in differentiated cells like hepatocytes and the hepatoma cell line H4IIE. Demethylation by 5-Aza-2-deoxycytidine was sufficient to induce Nestin in H4IIE cells. In quiescent stellate cells and embryonic stem cells, the Nestin expression was suppressed by histone H3 methylation at lysine 9, which is another epigenetic mechanism. Apart from the known induction of Nestin in cultured HSC, this intermediate filament protein was also induced after partial hepatectomy, indicating activation of HSC during liver regeneration. Taken together, this study demonstrates for the first time that the expression of stem cell-associated factors such as CD133, Notch1, and Notch3 is controlled by DNA methylation in HSC. The regulation of Nestin by DNA methylation seems to be restricted to differentiated cells, whereas undifferentiated cells use different epigenetic mechanisms such as histone H3 methylation to control Nestin expression.

Cancer-germline (CG) genes are a particular group of germline-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In a wide variety of tumors, the promoter of these genes is demethylated, and their transcription is activated. The mechanism underlying this tumor-specific activation is still unclear. It was recently suggested that CG gene expression may be a hallmark of stem cells, and that expression of these genes in several tumors may reflect the expansion of constitutively expressing cancer stem cells. To clarify this issue, we carefully evaluated the expression of several CG genes in human stem cells of embryonic and adult origin. We found no or very weak expression of CG genes in these cells. Consistently, the promoter of CG genes was highly methylated in these cells. We conclude that CG genes do not qualify as ''stemness'' genes, and propose that their activation in cancers results from a tumor-specific activation process. STEM CELLS 2009;27:822-824 Disclosure of potential conflicts of interest is found at the end of this article.