A new class of reversible cell cycle inhibitors

Abstract

The effects of three compounds on the cell cycle of HL-60 promyeloid leukemia cells has been examined. Ciclopirox olamine, an antifungal agent, and the compound Hoechst 768159 reversibly block the cell cycle at a point occurring roughly 1 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase OL activity, which acts in early S phase. Similar results are also obtained with the compound mimosine, a plant amino acid. Based on these data, it is concluded that all three agents inhibit cell cycle traverse at or very near the GUS phase boundary and identify a previously undefined reversible cell cycle arrest point. Key terms: Flow cytometry, G1 phase block, mimosine, ciclopirox olamineThe elucidation of the biochemical events that regulate the induction of DNA synthesis in mammalian cells is of fundamental importance to an understanding of cellular growth control. In particular, the transcriptional and posttranscriptional events that control the G1 to S phase transition have been the subject of intensive research in recent years. It has been demonstrated that the expression of a number of genes required for DNA synthesis increases in the late G1 or early S phase. These include thymidine kinase (6,28), dihydrofolate reductase (121, and DNA polymerase a (33). In addition, the posttranslational modification of several proteins occurs prior to or at the GUS border. The phosphorylation of the 34 kDa protein (p34) encoded by the mammalian cdc2 gene occurs in late Gli early S phase in serum-stimulated mouse 3T3 fibroblasts (20,23). A change in the phosphorylation state of the product of the retinoblastoma susceptibility locus, which may also be important in cell cycle progression, also occurs near the GUS boundary (2,4,9,22).Compounds that perturb the cell cycle a t or near the GUS phase boundary will be essential in determining the role of these different biochemical events in the induction cells of DNA synthesis. The resolution of physical techniques such as cell sorting and centrifugal elutriation is not sufficient to subdivide adequately the late Gliearly S phase on the basis of DNA content or cell volume and density. Although a number of agents have been described that reversibly block cell cycle progression, most of these act in S phase. These include aphidicolin (APH), which blocks DNA polymerase c1 activity (13); 5-fluorodeoxyuridine, which inhibits thymidylate synthase (15); and hydroxyurea, which acts on ribonucleotide reductase (27). Of these S phasespecific inhibitors, APH has been shown to act earliest in S phase (29). APH inhibits DNA replication by interfering with the binding of deoxynucleotide triphosphates to DNA polymerase (13). Both molecular and kinetic (1,7,10,11,21,26,29,31,34) studies indicate that APH blocks cell cycle progression in S phase cells that are undergoing active DNA replication and prevents the entry of cells into S phase just beyond the GliS border. The cloning and characterization of the eukaryotic chromosomal replication origins, which are DNA sequences associated w...

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis

“…As mentioned earlier, both HU and APH are widely used to arrest cells in S-phase to obtain cell populations synchronized in the cell cycle for a variety of biochemical or molecular studies (2,3,13,14). The present data clearly demonstrate that the cells treated with these inhibitors have a multiplicity of DSBs.…”

“…APH has also been applied to transiently arrest cells in S phase to obtain populations of cells synchronized in the cell cycle (13,14). As in the case of HU, inhibition of DNA replication by APH leads to DNA damage (5,(15)(16)(17).…”

Transcriptional Regulation of p21/CIP1 Cell Cycle Inhibitor by PDEF Controls Cell Proliferation and Mammary Tumor Progression

“…3A, 24-h mimosine treatment effectively synchronized both the PDEF 21 and the vector control cells at the G 0 /G 1 phase of the cell cycle with over 85% of each group being assigned to the G 0 /G 1 phase. A number of reports have successfully shown the reversibility of this treatment in the examination of cell cycle events (33)(34)(35)(36). Although the vector control cells had progressed out of the G 0 /G 1 phase and into the other phases of the cycle after 15 h, a significant portion (78%) of the PDEF 21 cells remained in the G 0 /G 1 phase at this time point.…”

Roles of cyclins A and E in induction of centrosome amplification in p53-compromised cells

“…We thus examined whether centrosome would reduplicate in the p53-inactivated cyclin E À/À MEFs upon exposure to Aph. Cyclins E þ / þ and E À/À MEFs expressing DNp53 were treated with Aph for 96 h. As expected (Hoffman et al, 1991), the majority of cells exposed to Aph were arrested at the G 1 /S boundary and S phase. We also found that endogenous cyclin A was upregulated in both cyclins E þ / þ and E À/À upon Aph treatment ( Figure 5A).…”