cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3␤ could inhibit nuclear import. Here, we report that GSK-3␤-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3␤, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division. During G1 phase, the D-type cyclins (D1, D2, D3) accumulate and assemble with either cyclin-dependent kinase 4 or 6 (CDK4 or CDK6) in response to mitogenic growth factors. The active cyclin D holoenzyme promotes G1 progression by inactivating the growth-suppressive properties of the retinoblastoma protein (Rb) through site-specific phosphorylation and by virtue of its ability to titrate CDK inhibitors such as p27Kip1 and p21Cip1 (Weinberg 1995;Sherr and Roberts 1999). Titration of p27Kip1 and p21 Cip1, in turn, facilitates activation of the cyclin E/CDK2 complex and subsequent entry and progression through the DNA synthetic (S) phase of the cell cycle. Although p27Kip1 and p21 Cip1 are effective inhibitors of cyclin E/CDK2 and cyclin A/CDK2 complexes, recent evidence demonstrates that they promote the assembly of cyclin D/CDK complexes (LaBaer et al. 1997) and are found in catalytically active cyclin D/CDK complexes in vivo (Cheng et al. 1999).Extracellular mitogens promote cellular proliferation via receptor-mediated signaling circuitry that ultimately converges on the cell cycle machine, and the D-type cyclins function as critical sensors of these signals. For example, receptor-dependent activation of Ras promotes the accumulation of active cyclin D1/CDK4 complexes via at least two pathways. First, activated Ras promotes transcription of the cyclin D1 gene through a kinase cascade involving Raf1, mitogen-activated protein kinase kinase (MEK), and the extracellular signal-regulated protein kinases (Albanese et al. 1995;Aktas et al. 1997;Lavoie et al. 1996;Winston et al. 1996;Kerkhoff and Rapp 1997;Weber et al. 1997). Assembly and activation of the cyclin D1/CDK4 holoenzyme are also augmented by this same pathway . Second, the rate of cyclin D1 proteasomal degradation is mediated by GSK-3␤-dependent phosphorylation of a single threonine residue (Thr-286) near the C terminus of cyclin D1 (Diehl et al. 1998). Mitogens inactivate GSK-3␤ via a pathway involving Ras, phosphatidylinositol 3-kinase, and protein kinase B/Akt (Rodriguez-Viciana et al.

There is increasing evidence that p21Cip1 and p27 Kip1are requisite positive regulators of cyclin D1⅐CDK4 assembly and nuclear accumulation. Both Cip and Kip proteins can promote nuclear accumulation of cyclin D1, but the underlying mechanism has not been elucidated. We now provide evidence that p21 Cip1 promotes the nuclear accumulation of cyclin D1 complexes via inhibition of cyclin D1 nuclear export. In vivo, we demonstrate that p21Cip1 can inhibit glycogen synthase kinase 3␤-triggered cyclin D1 nuclear export and phosphorylation-dependent nucleocytoplasmic shuttling. Furthermore, we find that cyclin D1 nuclear accumulation in p21/p27 null cells can be restored through inhibition of CRM1-depenendent nuclear export. The ability of p21Cip1 to inhibit cyclin D1 nuclear export correlates with its ability to bind to Thr-286-phosphorylated cyclin D1 and thereby prevents cyclin D1⅐CRM1 association.Cell cycle progression requires the sequential and ordered activation of the cyclin-dependent kinases (CDKs) 1 and inactivation of CDK inhibitors. D-type cyclins (D1, D2, D3), the regulatory subunit of the CDK4/6 kinase, function as critical mitogenic sensors that integrate growth factor-initiated signals with G 1 -phase progression (1). Mitogenic stimuli trigger the accumulation of active cyclin D1⅐CDK4 complexes through both increased cyclin expression and decreased cyclin proteolysis and through the promotion of cyclin D⅐CDK4 assembly (1). Mitogen-dependent expression of cyclin D1 depends upon growth factor-mediated activation of a signal transduction cascade consisting of Ras, Raf-1, and the extracellular signalregulated protein kinases (ERK1 and 2) (2-8). Accumulation of cyclin D1 during G 1 also relies upon mitogen-dependent inhibition of glycogen synthase kinase 3␤ (GSK-3␤) via activation of phosphatidylinositol 3-kinase and Akt (protein kinase B) (9). The subcellular localization of cyclin D1 complexes also oscillates during the cell cycle, being nuclear throughout G 1 -phase and cytoplasmic during the remainder of interphase (9 -11). Nuclear export of cyclin D1 is a major determinant of cyclin D1⅐CDK4 localization (12). Phosphorylation of cyclin D1 at a single threonine residue, Thr-286, by GSK-3␤ facilitates the binding of cyclin D1 with the nuclear exportin, CRM1, and thereby promotes cyclin D1 nuclear export (12). Because neither cyclin D1 nor CDK4 has a recognizable nuclear localization signal, the mechanisms governing cyclin D1 nuclear import remain undefined.

Mdm2 directly regulates the p53 tumor suppressor. However, Mdm2 also has p53-independent activities, and the pathways that mediate these functions are unresolved. Here we report the identification of a specific association of Mdm2 with Mre11, Nbs1, and Rad50, a DNA double strand break repair complex. Mdm2 bound to the Mre11-Nbs1-Rad50 complex in primary cells and in cells containing inactivated p53 or p14/p19 ARF , a regulator of Mdm2. Further analysis revealed that Mdm2 directly bound to Nbs1 but not to Mre11 or Rad50. Amino acids 198 -314 of Mdm2 were required for Mdm2/Nbs1 association, and neither the N terminus forkhead-associated and breast cancer C-terminal domains nor the C terminus Mre11 binding domain of Nbs1 mediated the interaction of Nbs1 with Mdm2. Mdm2 co-localized with Nbs1 to sites of DNA damage following ␥-irradiation. Notably, Mdm2 overexpression inhibited DNA double strand break repair, and this was independent of p53 and ARF, the alternative reading frame of the Ink4alocus. The delay in DNA repair imposed by Mdm2 required the Nbs1 binding domain of Mdm2, but the ubiquitin ligase domain in Mdm2 was dispensable. Therefore, Nbs1 is a novel p53-independent Mdm2 binding protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.

Mdm2 harnesses the p53 tumor suppressor, yet loss of one Mdm2 allele in Mdm2 +/± mice has heretofore not been shown to impair tumor development. Here we report that Mdm2 haplo-insuf®ciency profoundly suppresses lymphomagenesis in Em-myc transgenic mice. Mdm2 +/± Em-myc transgenics had greatly protracted rates of B cell lymphoma development with life spans twice that of wild-type transgenic littermates. Impaired lymphoma development was associated with drastic reductions in peripheral B cell numbers in Mdm2 +/± Em-myc transgenics, and primary pre-B cells from Mdm2 +/± Em-myc transgenics and Mdm2 +/± littermates were extremely susceptible to spontaneous apoptosis. Loss of p53 rescued all of the effects of Mdm2 haplo-insuf®ciency, indicating they were p53 dependent. Furthermore, half of the lymphomas that ultimately emerged in Mdm2 +/± Em-myc transgenics harbored inactivating mutations in p53, and the majority overcame haplo-insuf®ciency by overexpressing Mdm2. These results support the concept that Mdm2 functions are rate limiting in lymphomagenesis and that targeting Mdm2 will enhance p53-mediated apoptosis, compromising tumor development and/or maintenance.

BackgroundThe enumeration and characterization of circulating tumor cells (CTCs) in the blood of cancer patients is useful for cancer prognostic and treatment monitoring purposes. The number of CTCs present in patient blood is very low; thus, robust technologies have been developed to enumerate and characterize CTCs in patient blood samples. One of the challenges to the clinical utility of CTCs is their inherent fragility, which makes these cells very unstable during transportation and storage of blood samples. In this study we investigated Cell-Free DNA BCT™ (BCT), a blood collection device, which stabilizes blood cells in a blood sample at room temperature (RT) for its ability to stabilize CTCs at RT for an extended period of time.MethodsBlood was drawn from each donor into K3EDTA tube, CellSave tube and BCT. Samples were then spiked with breast cancer cells (MCF-7), transported and stored at RT. Spiked cancer cells were counted using the Veridex CellSearch™ system on days 1 and 4. The effect of storage on the stability of proteins and nucleic acids in the spiked cells isolated from K3EDTA tube and BCT was determined using fluorescence staining and confocal laser scanning microscopy.ResultsMCF-7 cell recovery significantly dropped when transported and stored in K3EDTA tubes. However, in blood collected into CellSave tubes and BCTs, the MCF-7 cell count was stable up to 4 days at RT. Epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) in MCF-7 cells isolated from BCTs was stable at RT for up to 4 days, whereas in MCF-7 cells isolated from K3EDTA blood showed reduced EpCAM and CK protein expression. Similarly, BCTs stabilized c-fos and cyclin D1 mRNAs as compared to K3EDTA tubes.ConclusionCell-Free DNA™ BCT blood collection device preserves and stabilizes CTCs in blood samples for at least 4 days at RT. This technology may facilitate the development of new non-invasive diagnostic and prognostic methodologies for CTC enumeration as well as characterization.

The tumor suppressor p19 ARF inhibits Mdm2, which restricts the activity of p53. Complicated feedback and control mechanisms regulate ARF, Mdm2, and p53 interactions. Here we report that ARF haploinsufficiency completely rescued the p53-dependent effects of Mdm2 haploinsufficiency on B-cell development, survival, and transformation. In contrast to Mdm2 þ /À B cells, Mdm2 þ /À B cells deficient in ARF were similar to wild-type B cells in their rates of growth and apoptosis and activation of p53. Consequently, the profoundly reduced numbers of B cells in Mdm2 þ /À El-myc transgenic mice were restored to normal levels in ARF þ /À Mdm2 þ /À El-myc transgenics. Additionally, ARF þ /À Mdm2 þ /À El-myc transgenics developed lymphomas at rates analogous to those observed for wild-type El-myc transgenics, demonstrating that loss of one allele of ARF rescued the protracted lymphoma latency in Mdm2 þ /À El-myc transgenics. Importantly, in ARF þ /À Mdm2 þ /À El-myc transgenic lymphomas, p53 was inactivated at the frequency observed in lymphomas of wild-type El-myc transgenics. Collectively, these results support a model whereby the stoichiometry of Mdm2 and ARF controls apoptosis and tumor development, which should have significant implications in the treatment of malignancies that have inactivated ARF.

BackgroundMessenger RNA (mRNA) expression levels in blood cells are important in disease diagnosis, prognosis and biomarker discovery research. Accurate measurements of intracellular mRNA levels in blood cells depend upon several pre-analytical factors, including delays in RNA extraction from blood after phlebotomy. Dramatic changes in mRNA expression levels caused by delays in blood sample processing may render such samples unsuitable for gene expression analysis.ObjectivesThis study was conducted to evaluate a blood collection tube, cell-free RNA-BCT® (RNA-BCT), for its ability to stabilize mRNA expression level in blood cells post-phlebotomy using indicator mRNAs in reverse transcription quantitative real-time PCR (RT-qPCR) assays.MethodsBlood samples from presumed healthy donors were drawn into both RNA-BCT and K3EDTA tubes and maintained at room temperature (18–22 °C). The samples were processed to obtain white blood cells (WBCs) at days 0, 1, 2 and 3. Total cellular RNA was extracted from WBCs and mRNA concentrations were quantified by RT-qPCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-fos, and p53 transcripts.ResultsWhile blood cells isolated from K3EDTA tubes showed significant changes in cellular mRNA concentrations for GAPDH, c-fos, and p53, these mRNAs concentrations were stable in blood drawn into RNA-BCT.ConclusionThe reagent in the RNA-BCT device stabilizes cellular mRNA concentrations for GAPDH, c-fos and p53 for at least three days at room temperature.