Showing papers by "Sally Kornbluth published in 2007"

Mcm10 and And-1/CTF4 recruit DNA polymerase α to chromatin for initiation of DNA replication

Abstract: The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase–polymerase complex, DNA polymerase (pol ), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol . And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol , and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10–And-1 interaction interferes with the loading of And-1 and of pol , inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol –primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery. The discovery also adds to the connection between replication initiation and sister chromatid cohesion.

Cdc25 and Wee1: analogous opposites?

Abstract: Movement through the cell cycle is controlled by the temporally and spatially ordered activation of cyclin-dependent kinases paired with their respective cyclin binding partners. Cell cycle events occur in a stepwise fashion and are monitored by molecular surveillance systems to ensure that each cell cycle process is appropriately completed before subsequent events are initiated. Cells prevent entry into mitosis while DNA replication is ongoing, or if DNA is damaged, via checkpoint mechanisms that inhibit the activators and activate the inhibitors of mitosis, Cdc25 and Wee1, respectively. Once DNA replication has been faithfully completed, Cdc2/Cyclin B is swiftly activated for a timely transition from interphase into mitosis. This sharp transition is propagated through both positive and negative feedback loops that impinge upon Cdc25 and Wee1 to ensure that Cdc2/Cyclin B is fully activated. Recent reports from a number of laboratories have revealed a remarkably complex network of kinases and phosphatases that coordinately control Cdc25 and Wee1, thereby precisely regulating the transition into mitosis. Although not all factors that inhibit Cdc25 have been shown to activate Wee1 and vice versa, a number of regulatory modules are clearly shared in common. Thus, studies on either the Cdc25 or Wee1-regulatory arm of the mitotic control pathway should continue to shed light on how both arms are coordinated to smoothly regulate mitotic entry.

HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53

Abstract: In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53.

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues

Abstract: Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.

Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine

Abstract: CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.

Abstract: Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

DNA replication and progression through the cell cycle.

Abstract: Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.