Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

Background: The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of gene

Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

DNA replication and the GINS complex: localization on extended chromatin fibers

<p>Abstract</p> <p>Background</p> <p>The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins.</p> <p>Results</p> <p>Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication.</p> <p>Conclusion</p> <p>In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.</p

Targeting acute myeloid leukemia by drug-induced c-MYB degradation

Despite advances in our understanding of the molecular basis for particular subtypes of acute myeloid leukemia (AML), effective therapy remains a challenge for many individuals suffering from this disease. A significant proportion of both pediatric and adult AML patients cannot be cured and since the upper limits of chemotherapy intensification have been reached, there is an urgent need for novel therapeutic approaches. The transcription factor c-MYB has been shown to play a central role in the development and progression of AML driven by several different oncogenes, including mixed lineage leukemia (MLL)-fusion genes. Here, we have used a c-MYB gene expression signature from MLL-rearranged AML to probe the Connectivity Map database and identified mebendazole as a c-MYB targeting drug. Mebendazole induces c-MYB degradation via the proteasome by interfering with the heat shock protein 70 (HSP70) chaperone system. Transient exposure to mebendazole is sufficient to inhibit colony formation by AML cells, but not normal cord blood-derived cells. Furthermore, mebendazole is effective at impairing AML progression in vivo in mouse xenotransplantation experiments. In the context of widespread human use of mebendazole, our data indicate that mebendazole-induced c-MYB degradation represents a safe and novel therapeutic approach for AML

A new monoclonal antibody against DNA ligase I is a suitable marker of cell proliferation in cultured cell and tissue section samples

The extensive characterization of the replicative human DNA ligase I (LigI) undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation.We have produced a new monoclonal antibody (5H5) against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded) demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry

A degradation-resistant c-Myb mutant cooperates with Bcl-2 in enhancing proliferative potential and survival of hematopoietic cells

The c-myb gene is preferentially expressed in primitive hematopoietic cell and plays a central role in the control of cell proliferation, differentiation and survival by regulating the transcription of several genes implicated in these processes including the antiapoptotic Bcl-2. We show here that, compared to wild-type c-Myb, overexpression of a degradation resistant c-Myb mutant [Delta(358-452) c-Myb] enhances the clonogenic potential of hematopoietic progenitors as indicated by increased cytokine-dependent primary and secondary colony formation of Lin(-) Sca-1(+) Kit(+) mouse marrow cells. Moreover, proliferation assays of IL-3 dependent myeloid precursor 32Dcl3 cells co-expressing Bcl-2 and c-Myb indicate that these cells continue to proliferate in the absence of IL-3 and this effect is more apparent in cells expressing the degradation resistant Delta(358-452) c-Myb. Interestingly, overexpression of Delta(358-452) c-Myb is by itself sufficient to protect 32Dcl3 cells from apoptosis induced by IL-3 deprivation; moreover, these cells are also increased in number which most likely reflects the enhanced proliferative potential conferred by Delta(358-452) c-Myb to apoptosis-resistant cell

Transcriptional repression of c-Myb and GATA-2 is involved in the biologic effects of C/EBPalpha in p210BCR/ABL-expressing cells

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets