The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RAR␣), and PLZF-RAR␣ encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RAR␣, or PLZF-RAR␣. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (␥-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF-and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. ␤-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and ␥-irradiation which was mediated by p53. cyclin A1 ؊/؊ cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1؊/؊ cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.The cell cycle is regulated by external signals, especially in multicellular organisms, where cell proliferation is central to growth and differentiation (34). Internal signals ensure that cell cycle transitions do not occur until all required molecular events have been completed (20). The internal signals become evident as cell cycle checkpoints upon an insult to the cell (e.g., DNA damage) which arrests the cell cycle while repair occurs (10,14). Both internal and external signals modulate the activity of the cyclin-dependent kinases (CDKs) that catalyze the ordered transitions from one phase of the cell cycle to the next. Cyclin E and cyclin A2 both associate with CDK2, and cyclin A2 is essential for DNA replication and proliferation in somatic cells (8). Embryos with a homozygous deletion of cyclin A2 are not capable of proliferative growth (28). During G 1 and S phases, the kinase activity of CDK2 associates first with cyclin E and later with cyclin A2. The timing of these processes as well as the appropriate concentration of cyclin/CDK2 complexes is crucial for successful DNA replication (8).Cyclin A1 is a second A-type cyclin that binds CDK2. Cyclin A1 is abundantly expressed in the testis and has previously been shown to be essential for entry into the metaphase of meiosis I in the male germ line in mice (38, 44). Cyclin A1 is expressed at low levels in most other tissues, but no phenotype other than male infertility has been reported for mice lacking the cyclin A1 gene (19,42). The expression of cyclin A1 in hematopoietic progenitor cells and in acute myeloid leukemia is best characterized in somatic cells (46). Surprisingly, recent microarray data suggested that cyclin A1 was transcriptionally induced following p53 activation (21).Thus, the physiological role of cyclin A1 in somatic cells remains unknown. This prompted us to analyze the mechanisms of cyclin A1 induction in somatic cel...

Suppression of collagen synthesis is a major therapeutic goal in the treatment of fibrotic disorders. We show here that ␣-melanocyte-stimulating hormone (␣-MSH), a neuropeptide well known for its pigment-inducing capacity, modulates collagen synthesis and deposition. ␣-MSH in vitro suppresses the synthesis of collagen types I, III, and V and down-regulates the secretion of procollagen type I C-terminal peptide (PICP) in human dermal fibroblasts treated with the fibrogenic cytokine transforming growth factor-␤ 1 (TGF-␤ 1 ). ␣-MSH did not interfere with TGF-␤ 1 signaling, because TGF-␤ 1 -induced expression of collagen mRNA was not affected, implying a posttranscriptional mechanism. Human dermal fibroblasts in vitro express a high affinity binding site for MSH, which was identified by reverse transcription PCR and immunofluorescence analysis as the melanocortin-1 receptor (MC-1R). Immunohistochemical studies on normal adult human skin confirmed MC-1R expression in distinct dermal fibroblastic cells. The MC-1R on fibroblasts appears to be functionally relevant because ␣-MSH increased the amount of intracellular cAMP, and coincubation with a synthetic peptide corresponding to the human Agouti signaling protein abrogated the inhibition of TGF-␤ 1 -induced PICP secretion by ␣-MSH. To assess the in vivo relevance of these findings, a mouse model was used in which dermal fibrosis was induced by repetitive intracutaneous injections with TGF-␤ 1 . The inductive activity of TGF-␤ 1 on collagen deposition and the number of dermal cells immunoreactive for vimentin and ␣-smooth muscle actin was significantly suppressed by injection of ␣-MSH.

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1 À/À cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1 À/À mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.