Millipore Sigma Vibrant Logo
 

05636mm


194 Results Advanced Search  
Showing
Products (0)
Documents (194)
Site Content (0)

Narrow Your Results Use the filters below to refine your search

Document Type

  • (166)
  • (19)
  • (9)
Can't Find What You're Looking For?
Contact Customer Service

 

  • Molecular basis for G2 arrest induced by 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine and consequences of checkpoint abrogation. 16061671

    2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by beta-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. 20101220

    DNA double-stranded breaks (DSBs) elicit a checkpoint response that causes a delay in cell cycle progression. Early in the checkpoint response, histone H2AX is phosphorylated in the chromatin region flanking the DSB by ATM/ATR and DNA-PK kinases. The resulting foci of phosphorylated H2AX (gamma-H2AX) serve as a platform for recruitment and retention of additional components of the checkpoint-signaling cascade that enhance checkpoint signaling and DSB repair. Upon repair, both the assembled protein complexes and the chromatin modifications are removed to quench the checkpoint signal. In this study, we show that the DNA damage-responsive Wip1 phosphatase is bound to chromatin. Moreover, Wip1 directly dephosphorylates gamma-H2AX and cells depleted of Wip1 fail to dephosphorylate gamma-H2AX during checkpoint recovery. Conversely, premature activation of Wip1 leads to displacement of MDC1 from damage foci and prevents activation of the checkpoint. Taken together, our data show that Wip1 has an essential role in dephosphorylation of gamma-H2AX to silence the checkpoint and restore chromatin structure once DNA damage is repaired.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • MDC1 cleavage by caspase-3: a novel mechanism for inactivating the DNA damage response during apoptosis. 21148072

    Recently, we identified the "apoptotic ring," containing phosphorylated histone H2AX (γ-H2AX), as an early chromatin modification during apoptosis. Because γ-H2AX initiates the DNA damage response (DDR), we tested whether the apoptotic H2AX response leads to the full recruitment of the DDR factors that normally coordinate DNA repair and cell-cycle checkpoints. We show that the apoptotic H2AX response does not recruit the DDR factors because MDC1 (mediator of DNA damage checkpoint protein 1), which normally binds to γ-H2AX in response to DNA damage and amplifies the DDR, is cleaved by caspase-3. This cleavage separates the BRCT and FHA domains of MDC1 and constitutes a novel mechanism for the inactivation of DNA repair in apoptotic cells. Also, we show that downregulation of MDC1 increases the apoptotic response to TRAIL. Together, these results implicate MDC1 in the cellular apoptotic response.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M. 22837195

    Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Molecular mode of action and role of TP53 in the sensitivity to the novel epothilone sagopilone (ZK-EPO) in A549 non-small cell lung cancer cells. 21559393

    Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but--compared to high concentrations (40 nM)--only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • DNA-Dependent Protein Kinase and Ataxia Telangiectasia Mutated (ATM) Promote Cell Survival in Response to NK314, a Topoisomerase II{alpha} Inhibitor. 21546556

    4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα, leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314, to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314, and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA, a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs, as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit, Ku80, ataxia telangiectasia mutated (ATM), BRCA2, or XRCC3 compared with wild-type cells, indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore, both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo. 21423202

    Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence. 21841825

    Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Androgen depletion induces senescence in prostate cancer cells through down-regulation of Skp2. 21677876

    Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Histone lysine methyltransferase SETD8 promotes carcinogenesis by deregulating PCNA expression. 22556262

    Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple