Cellular effects of olomoucine, an inhibitor of cyclin‐dependent kinases

Abstract

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.

Cdk2-dependent phosphorylation of p27 facilitates its Myc-induced release from cyclin E/cdk2 complexes

“…Two experiments were set up to test this prediction. First, we tested whether a speci®c inhibitor of cdk2 kinase activity, roscovitine, interfered with Myc-induced loss of p27 from cyclin E/cdk2 complexes (Abraham et al, 1995;Vesely et al, 1994). RAT1-MycER cells were induced either in the absence or presence of roscovitine.…”

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R‐roscovitine)

“…10 Compounds of this class have been shown to block proliferation of various tumour types in cell culture. [11][12][13] Our study describes the relative potency of CYC202 (R-roscovitine) against purified recombinant CDKs and relates this result to in vivo growth inhibitory properties in a panel of human cancer cell lines representative of human solid tumours, and compares its cytotoxicity in proliferating tumour cells vs. non-proliferating cells. The effects of CYC202 on cell cycle distribution in Lovo colorectal carcinoma cells is discussed.…”

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation

“…No signal was observed in the negative control. To see whether phosphorylation of Fen1 was linked to Cdks activity, myc-tagged Fen1 was expressed in 293T cells treated during 24 h with 40 mm Olomoucine (a specific inhibitor of Cdk1 and Cdk2) (Abraham et al, 1995) dissolved in DMSO. Cells treated with DMSO alone were used as a negative control.…”