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Biomedical subjects

R Heald

Publications and source records attributed to R Heald.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis and biological evaluation of myoseverin derivatives: microtubule assembly inhibitors.

Myoseverin, a trisubstituted purine, inhibits microtubule assembly in vitro, interferes with normal mitotic spindle assembly, and arrests the cell cycle in mitosis in U937 cells. We synthesized a variety of myoseverin derivatives and screened them for inhibition of spindle assembly in Xenopus egg extracts and for microtubule disassembly in vitro. Selected compounds were tested against 60 cancer cell lines at the National Cancer Institute as possible anticancer drug candidates.

Animals↗
PubMed2001

Kinetochore function: the complications of becoming attached.

Kinetochores are specialized protein complexes assembled on centromeric DNA that connect to spindle microtubules and mediate proper chromosome segregation during cell division. Recent results have implicated specific kinetochore proteins in microtubule attachment.

Fungal Proteins↗
PubMed2001

Regulation of Op18 during spindle assembly in Xenopus egg extracts.

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.

Animals↗
PubMed2001

Chromosome movement: dynein-out at the kinetochore.

Cell biologists have long speculated that a minus end-directed motor localized at kinetochores contributes to the poleward movement of chromosomes during mitosis. Two recent studies provide direct evidence that cytoplasmic dynein can perform this function.

Animals↗
PubMed2001

Importin beta is a mitotic target of the small GTPase Ran in spindle assembly.

The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.

Animals↗
PubMed2001

Potent inhibition of telomerase by small-molecule pentacyclic acridines capable of interacting with G-quadruplexes.

A novel pentacyclic acridine, 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4), has been identified as a potent inhibitor of telomerase in the cell-free telomeric repeat amplification protocol (TRAP). Modeling and biophysical studies suggest that RHPS4 inhibits telomerase through stabilization of four-stranded G-quadruplex structures formed by single-stranded telomeric DNA. In contrast to G-quadruplex interactive telomerase inhibitors described previously, RHPS4 inhibited telomerase at submicromolar levels (50% inhibition in the TRAP assay at 0.33 +/- 0.13 microM). Moreover, RHPS4 exhibited a wide differential between this potent inhibition of telomerase and acute cellular cytotoxicity (mean IC(50) value of 7.02 microM in 4-day growth inhibition assay). RHPS4, when added to 21NT breast cancer cells at nonacute cytotoxic concentrations (200 nM) every 3 to 4 days, induced a marked cessation in cell growth after 15 days. Similar effects were observed using another cell line possessing relatively short telomeres, A431 human vulval carcinoma cells, but not in a human ovarian carcinoma cell line (SKOV-3) possessing relatively long telomeres. In 21NT cells, growth cessation was accompanied by an increase in cells in the G(2)/M phase of the cell cycle, a reduction in cellular telomerase activity, and a lower expression of the hTERT gene. These effects occurred in the absence of a detectable reduction in telomere length as measured by slot blotting. RHPS4 also induced a cessation of growth of GM847 cells that maintain telomeres by a nontelomerase alternative mechanism for lengthening telomeres (ALT) after 15 days. RHPS4 represents a promising G-quadruplex interactive small molecule that is a potent cell-free inhibitor of human telomerase and induces growth inhibitory effects in human tumor cell lines after prolonged (2-week) exposure to nonacute cytotoxic drug concentrations.

DNA↗
PubMed2000

Formation of spindle poles by dynein/dynactin-dependent transport of NuMA.

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

Biological Transport↗
PubMed2000

Spindles get the ran around.

Despite its fundamental role in cell division, the mitotic spindle remains an enigmatic figure in cell biology. This is due to the complex dynamic behaviour of microtubules, which form the spindle fibres responsible for segregating chromosomes to opposite ends of the cell during mitosis. Recent reports indicate that the small GTPase Ran, which plays a key role in nuclear transport, also has a role in mitosis by regulating microtubule nucleation and/or growth. The race is now on to determine how Ran exerts its effects on spindle assembly.

Animals↗
PubMed1999

A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retained the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquired phenotypic characteristics of differentiated macrophages and became arrested in the cell cycle with a 4N DNA content. AP also inhibited mitosis in Xenopus egg extracts, suggesting that it acted on an evolutionarily conserved cell cycle regulatory pathway. Affinity chromatography and biochemical reconstitution experiments with Xenopus egg extracts identified cyclin-dependent kinase (CDK) 1-cyclin B as a target of the compound. Although AP potently inhibited immunoprecipitates of both human CDK1 and CDK2 from human leukemic cell extracts, our results indicate that the compound preferentially targets the G2/M-phase transition in vivo.

Animals↗
PubMed1999

Microtubule-based motor function in mitosis.

Microtubule-based motors are essential both for the proper assembly of the mitotic spindle and for chromosome segregation. Mitotic motors in the yeast Saccharomyces cerevisiae exhibit either overlapping or opposing activities in order to achieve proper spindle function, whereas the analysis of motors using vertebrate cytoplasmic extracts has revealed less functional redundancy. In several systems, biochemical, genetic and two-hybrid approaches have been used both to identify associated nonmotor proteins and to address the molecular mechanisms behind kinetochore movements during chromosome alignment and segregation.

Animals↗
PubMed1997

Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization.

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end-directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

Animals↗
PubMed1997

Role of chromosomes in assembly of meiotic and mitotic spindles.

The assembly of a mitotic spindle requires the interaction of microtubules with chromosomes. As a cell enters mitosis, long microtubules are converted to short ones, as microtubules become unstable. Dynamic microtubules are then stabilised by chromosomes, forming a bipolar spindle. In this review, we discuss the different roles of kinetochores and chromosome arms during spindle assembly. Kinetochores, required for proper chromosomes segregation, capture microtubules and maintain attachment. Chromosome arms greatly enhance microtubule stability, and alone can be sufficient for spindle assembly.

Animals↗
PubMed1996

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.

Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.

Animals↗
PubMed1993

Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase.

The wee1 tyrosine kinase and cdc25 tyrosine phosphatase of fission yeast play antagonistic roles in the induction of mitosis through cdc2 regulation. We show here that the human wee1-like tyrosine kinase is a nuclear protein that ensures the completion of DNA replication prior to mitosis in cells expressing otherwise catastrophic levels of cdc2 activators. Paradoxically, wee1-rescued cells display very high levels of mitotic cdc2 kinase activity. We account for this anomaly by our observation that the cdc2 activator, cdc25C, is a cytoplasmic protein that, like cyclin B1, enters the nucleus at the G2/M transition. Thus, cdc2 is likely to be activated in the cytoplasm and requires nuclear localization to initiate both cytoplasmic and nuclear mitotic transformations. The human wee1 kinase appears to coordinate the transition between DNA replication and mitosis by protecting the nucleus from this cytoplasmically activated cdc2 kinase.

Amino Acid Sequence↗