Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by expression of the p210-BCR-ABL fusion protein. We demonstrate in a murine model of p210-BCR-ABL-induced MPD that gene targeting of Rac1 and Rac2 significantly delays or abrogates disease development. Attenuation of the disease phenotype is associated with severely diminished p210-BCR-ABL-induced downstream signaling in primary hematopoietic cells. We utilize NSC23766, a small molecule antagonist of Rac activation, to validate biochemically and functionally Rac as a molecular target in both a relevant animal model and in primary human CML cells in vitro and in a xenograft model in vivo, including in Imatinib-resistant p210-BCR-ABL disease. These data demonstrate that Rac is an additional therapeutic target in p210-BCR-ABL-mediated MPD.

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G 1 and G 2 /M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53 22/23 . An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at ؊76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNAprotein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.Inactivation of p53 tumor suppressor gene occurs in over half of all human tumors, implying that loss of this gene represents a fundamentally important step in genomic instability and susceptibility to malignant transformation (1, 2). The underlying mechanism of tumor suppressor activity of p53 resides in part in its ability to bind DNA in a sequence-specific manner to activate gene transcription (2). It has been reported that a substantial number of genes containing the p53-binding site(s) are activated by p53. These include mdm2 (3, 4), p21/WAF-1 (5), Gadd45 (6), cyclin G (7), bax (8), and an insulin-like growth factor-binding protein (IGF-BP3) (9). p21 and Gadd45 were implicated in the p53-mediated cell cycle regulation (10, 11), while bax and IGF-BP3 were involved in the induction of apoptosis (8,9).In addition to playing a role as a DNA-binding dependent transcription activator, p53 has also been reported to negatively regulate the transcription of a number of genes. These genes include presenilin 1 (12), topoisomerase II␣ (13, 14), map4 (15), O 6 -methylguanine-DNA methyltransferase (16), insulin receptor (17), mdr-1 (18), hsp70 1 (19), interleukin-6 (20), bcl2 (21), c-fos (22), and other viral and cellular promoters (23). In contrast to the transcription activation by p53, no consensus sequence has been found in the promoters that are repressed by p53. It was initially reported that only the promoters containing a TATA box, but not those containing an initiator element, are repressed by p53 (24). This finding...

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While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine-tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl-DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR-302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2-mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell-based therapies.