Showing papers by "David Beach published in 1992"

Growth-regulated expression of D-type cyclin genes in human diploid fibroblasts.

Abstract: The human CCND1 cyclin D1/PRAD1 gene was previously identified by a genetic screen for G1 cyclin function in Saccharomyces cerevisiae and also was identified as the putative BCL1 oncogene. However, its role in human cell proliferation is not known. To determine if expression of human D-type cyclin genes correlates with the state of cell growth, we examined the level of mRNAs for CCND1 and a related gene, CCND3, in normal human diploid fibroblasts (HDF). The levels of both mRNAs decrease upon serum depletion or at high cell densities. Following stimulation of quiescent fibroblasts with serum, the mRNA levels increase gradually to a peak at about 12 hr, prior to the onset of S phase. Induction of cyclin gene expression by serum is reduced concomitantly with the decline in FOS induction in aging HDFs, suggesting a possible relationship to the decrease in the proliferative response to mitogens during cellular senescence. Cycloheximide partially blocks the induction of CCND1 and CCND3 gene expression by serum, suggesting that both de novo protein synthesis-dependent and -independent pathways contribute to induction. Treatment of HDFs with defined growth factors suggests a correlation between CCND mRNA induction and DNA synthesis. However, induction of these genes is not sufficient for the transition from quiescence through G1 into S phase.

Human p53 inhibits growth in Schizosaccharomyces pombe.

Abstract: Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.

Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase.

Abstract: A universal intracellular factor, the > (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin B(cdc13). In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds. The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts in Escherichia coli and easily purified by affinity chromatography on glutathione-agarose. Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined. Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity. However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor. The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery.

D-type cyclin and uses related thereto

Abstract: A novel class of cyclins, referred to as D-type cyclins, of mammalian origin, particularly human origin, DNA and RNA encoding the novel cyclins, and a method of identifying other D-type and non-D type cyclins. Also disclosed are a method of detecting an increased level of a D-type cyclin and a method of inhibiting cell division by interfering with formation of the protein kinase-D type cyclin complex essential for cell cycle start.

NOVEL HUMAN cdc25 GENES, ENCODED PRODUCTS AND USES THEREFOR

Abstract: L'invention se rapporte a deux genes humains cdc25 non decrits precedemment et designes cdc25 A et cdc25 B, qui se sont reveles presenter une activite de tyrosine phosphatase endogene pouvant etre activee specifiquement par de la cycline de type B, en l'absence totale de cdc2 The invention relates to two human genes cdc25 not previously described and designated cdc25 cdc25 A and B, which were found to have an endogenous tyrosine phosphatase activity can be specifically activated by cyclin B, in the total absence of cdc2 Ces travaux permettent de considerer de nouvelles possiblites de regulation du cycle cellulaire dans des cellules eucaryotes et, particulierement, de regulation de l'activite de tyrosine phosphatases specifiques jouant un role capital dans le cycle cellulaire These works allow us to consider new regulatory possibilities of the cell cycle in eukaryotic cells and, especially, regulating the activity of tyrosine phosphatases specific playing a crucial role in the cell cycle L'invention se rapporte a des procedes de regulation du cycle cellulaire et, particulierement, de regulation de l'activation de la cdc2-kinase par l'intermediaire de la modification de l'activite et/ou des niveaux de tyrosine phosphatases ou par l'intermediaire de la modification de l'interaction des constituants de MPF The invention relates to methods of regulating the cell cycle and, especially, the activation control of cdc2 kinase via the change in the activity and / or tyrosine phosphatases levels or by means of changing the interaction of the constituents of MPF L'invention se rapporte egalement a des agents ou a des compositions efficaces dans ledit procede de regulation (inhibition ou activation) du cycle cellulaire The invention also relates to agents or compositions effective in said method for regulating (inhibition or activation) of the cell cycle Lesdits agents ou lesdites compositions peuvent etre des inhibiteurs (tels que des peptides ou des composes a poids molleculaire faible, soit organiques soit inorganiques) de l'activite catalytique des tyrosine proteine-phosphatases specifiques (particulierement cdc25), des agents bloquants agissant sur l'interaction ou sur la liaison de la tyrosine proteine-phosphatase specifique avec la cycline ou avec le complexe cycline/cdc2, ou bien des agents agissant directement sur l'activite catalytique des proteines phosphatases The agents said compositions or can be inhibitors (such as peptides or compounds molleculaire low weight, either inorganic or organic) of the catalytic activity of tyrosine specific protein phosphatases (particularly cdc25), blocking agents acting on the interaction or binding of the specific protein tyrosine phosphatase with cyclin or the cyclin / cdc2 or agents acting directly on the catalytic activity of protein phosphatases L'invention se rapporte egalement a un essai servant a identifier des agents qui modifient la stimulation de l'activite de kinase de pre-MPF et, de ce fait, modifient l'activation du facteur de promotion de phase M (MPF) et entrent dans la mitose The invention also relates to an assay for identifying agents that alter the stimulation of pre-MPF kinase activity and, thereby, alter the activation of the phase promoting factor M (MPF) and enter in mitosis Lesdits agents sont egalement objet de ladite invention The agents are also subject of the invention

Method of screening for antimitotic compounds using the CDC25 tyrosine phosphatase

Abstract: A method of identifying compounds or molecules which alter (enhance or inhibit) stimulation of kinase activity of pre-MPF and, thus, alter (enhance or inhibit) activation of MPF and entry into mitosis. The present method thus makes it possible to identify compounds or molecules which can be administered to regulate the cell cycle; such compounds are also the subject of this invention.