A new screening test for antimitotic compounds using the universal M phase-specific protein kinase, p34cdc2/cyclin Bcdc13, affinity-immobilized on p13suc1-coated microtitration plates.
A universal intracellular factor, the "M phase-Promoting Factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. This factor displays an easily assayable histone H1 kinase (H1K) activity and is composed of at least two subunits, p34cdc2 (catalytic) and cyclin Bcdc13 (regulatory). We describe here a microtitration plate assay using affinity-immobilized H1K-MPF as a cell cycle-specific target to screen for antimitotic compounds. First, meiotic starfish oocytes were selected as the most convenient and abundant source of M phase extracts containing high levels of H1K. Second, we used the strong and specific interaction between p34cdc2 and p13suc1 to affinity-immobilize H1K-MPF on p13suc1-coated microtitration plates. p13suc1-coated wells specifically retain the M phase kinase, the activity of which is assayed with histone H1 and gamma-32P-ATP. Among 10 microtitration plates, Maxisorp plates (Nunc) proved to be the most efficient at retaining H1K through p13suc1. Experimental conditions to coat the plates with p13suc1, to immobilize and to measure p34cdc2/cyclin Bcdc13 kinase activity, as well as to store p13suc1-precoated plates, have been optimized. Using this system we tested 18 currently used anticancer drugs and S or G2 inhibitors; none of them displayed any inhibitory activity. The microtitration assay has allowed the detection of two H1K inhibitors, isopentenyladenine (I50: 40 microM) and staurosporine (I50: 3.2 nM). This affinity-immobilized H1K-MPF can thus now be used as a simple screening system to detect inhibitors of a major cell cycle-regulating component. This method may prove useful to screen for antimitotic compounds of potential anticancer interest.
Rialet
V
V
CNRS-Station Biologique, Roscoff, France.
Meijer
L
L
D000109:Acetylcholine / Q000494:pharmacology; D000327:Adsorption; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005260:Female; D005306:Fertilization; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D008297:Male; D008938:Mitosis / Q000187:drug effects*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006656:Protamine Kinase / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D012617:Sea Urchins; D013215:Starfish
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase; D000109:Acetylcholine