aphidicolin and Leukemia--Promyelocytic--Acute

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Topics: 14-3-3 Proteins; Antineoplastic Agents; Aphidicolin; Asteraceae; Biological Products; Cell Cycle Checkpoints; Cell Death; Endophytes; Ergosterol; Fungi; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Inflammation; Leukemia, Promyelocytic, Acute; Microtubules; Mitochondria; Oxidative Stress; Proteome; Proteomics; rho Guanine Nucleotide Dissociation Inhibitor beta; Thymosin; Tubulin

We have studied changes in cyclin A- and B1-dependent kinases during apoptosis induced in human promyelocytic leukemia (HL60) cells treated with the topoisomerase I inhibitor camptothecin. We found that cyclin B1/Cdc2 kinase activity transiently increases within 30 min after camptothecin treatment. This increase is followed by a rapid inactivation of the cyclin B1/Cdc2 kinase that is associated with Cdc2 tyrosine phosphorylation without any change in Cdc2 or cyclin B1 protein levels. The DNA polymerase inhibitor aphidicolin abrogates camptothecin-induced changes in cyclin B1/Cdc2 kinase activity, indicating that DNA replication-induced DNA damage is essential for both Cdc2 alterations and apoptosis activation. Apoptosis and the initial cyclin B1/Cdc2 kinase activation were amplified using synchronized S-phase cells, and cyclin A/cdk2 kinase did not change under these conditions. The same transient activation and subsequent inactivation of cyclin B1/Cdc2 kinase were observed after DNA damage by etoposide or bis-(2-chloroethyl)methylamine hydrochloride. These observations suggest that DNA damage promotes the transient and unscheduled stimulation of cyclin B1/Cdc2 kinase activity in HL60 cells prior to apoptosis.

Topics: Aphidicolin; Apoptosis; Camptothecin; CDC2 Protein Kinase; Cyclins; DNA Damage; Enzyme Activation; Etoposide; Humans; Leukemia, Promyelocytic, Acute; Mechlorethamine; Phosphorylation; S Phase; Tumor Cells, Cultured

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca2+/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosome-sized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Aminoquinolines; Aphidicolin; Apoptosis; Benzamides; Calcium; Calmodulin; Camptothecin; Cell Cycle; Chromatin; Cyclosporine; DNA; Egtazic Acid; Ethers, Cyclic; Humans; Isoquinolines; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Piperazines; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Polycyclic Compounds; Protein Kinase C; Spermine; Staurosporine; Teniposide; Tetradecanoylphorbol Acetate; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Zinc

To search for possible synergy between topoisomerase (topo) II-directed chemotherapeutic agents and topo I-directed agents, IL-60 human progranulocytic leukemia cells were incubated with etoposide in the absence or presence of camptothecin (CPT). Treatment of HL-60 cells for 1 h with 15-20 microM etoposide resulted in the death of 99-99.9% of the cells as assessed by colony formation in soft agar. Unexpectedly, simultaneous incubation with 1 microM CPT increased the survival of etoposide-treated cells as much as 30-fold. Inhibition of etoposide cytotoxicity was observed at CPT concentrations as low as 0.01 microM and was one-half maximal at 0.1 microM. CPT also antagonized the cytotoxicity of 4'-(9-acridinylamino)methanesulfon-M-anisidide and daunorubicin, two structurally unrelated topo II-directed agents. Topotecan, a CPT analogue currently undergoing Phase I clinical trials, had a similar effect. Studies using an alkaline unwinding assay (to measure DNA strand breaks) and Western blotting (to assess formation of covalent adducts involving topo II) revealed that CPT did not alter the ability of etoposide to stabilize topo II-DNA adducts. CPT is a potent inhibitor of both DNA and RNA synthesis. To further assess the mechanism by which CPT diminished the cytotoxicity of topo II-directed agents, inhibitors of DNA synthesis or RNA synthesis were substituted for CPT. Aphidicolin, an inhibitor of replicative DNA polymerases, enhanced the survival of etoposide-treated HL-60 cells less than 3-fold. In contrast, inhibitors of RNA synthesis (cordycepin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) enhanced the survival of etoposide-treated HL-60 cells as much as 20-fold. The potential biological and therapeutic implications of these results are discussed.

Topics: Amsacrine; Aphidicolin; Blotting, Western; Camptothecin; Colony-Forming Units Assay; Cycloheximide; Daunorubicin; Deoxyadenosines; Dichlororibofuranosylbenzimidazole; Diterpenes; DNA Polymerase II; DNA Replication; Drug Synergism; Etoposide; Female; Humans; Leukemia, Promyelocytic, Acute; Mutagens; RNA; Topoisomerase II Inhibitors; Topotecan; Tumor Cells, Cultured

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 alpha 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 G1/S phase boundary and identify a previously undefined reversible cell cycle arrest point.

Topics: Antifungal Agents; Aphidicolin; Cell Cycle; Cell Line; Ciclopirox; Diterpenes; DNA Polymerase II; G1 Phase; Humans; Leukemia, Promyelocytic, Acute; Mimosine; Pyridones; S Phase; Thiazoles; Tumor Cells, Cultured

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective chemotherapeutic agent that incorporates into DNA and results in DNA fragmentation. Recent work has demonstrated that ara-C transiently induces expression of the c-jun immediate early response gene. The present studies in HL-60 myeloid leukemia cells extend these findings by demonstrating that the increase in c-jun mRNA levels at 6 h of ara-C treatment is regulated by a transcriptional mechanism. In contrast, the subsequent down-regulation of c-jun expression is controlled by a posttranscriptional decrease in the stability of the c-jun transcripts. Previous work in phorbol ester treated cells has indicated that c-jun expression is regulated by the activation of protein kinase C. The present results demonstrate that protein kinase C activity is increased in ara-C-treated cells. This increase was maximal at 60 min and remained detectable through 6 h of ara-C exposure. Moreover, the induction of c-jun transcripts by ara-C was inhibited by the isoquinolinesulfonamide derivative H7, but not by HA1004, suggesting that this effect is mediated by protein kinase C. Ara-C-induced c-jun expression was also inhibited by staurosporine, another inhibitor of protein kinase C. Taken together, these results indicate that the cellular response to ara-C includes the activation of protein kinase C and that ara-C potentially induces c-jun transcription by a protein kinase C dependent signaling mechanism.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aphidicolin; Cell Line; Cell Nucleus; Cycloheximide; Cytarabine; Dactinomycin; Diterpenes; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Kinetics; Leukemia, Promyelocytic, Acute; Piperazines; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-jun; Proto-Oncogenes; RNA, Neoplasm; Sulfonamides; Transcription Factors; Transcription, Genetic

We investigated the accumulation of DNA strand breaks in a human promyelocytic leukemia cell line, HL-60, treated with methotrexate (MTX) and 1-beta-D-arabinofuranosylcytosine (Ara-C). The sequential treatment with MTX then Ara-C had a synergistic effect on the formation of DNA strand breaks, which was dependent on MTX and Ara-C concentrations. On the other hand, when Ara-C preceded MTX, no such synergism was observed. The addition of both thymidine and hypoxanthine to this system, but not thymidine or hypoxanthine alone, abolished the synergism. Pretreatment with MTX augmented the generation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate. However, this augmentation did not necessarily correlate with the amount of strand breaks. Whatever the underlying mechanism of this synergism is, our present data provide one possible biochemical basis for sequential MTX and Ara-C therapy.

Topics: Aphidicolin; Cell Line; Cytarabine; Diterpenes; DNA Damage; Drug Combinations; Drug Synergism; Humans; Hypoxanthine; Hypoxanthines; Leucovorin; Leukemia, Promyelocytic, Acute; Methotrexate; Phosphorylation; Thymidine

Using a promyelocytic leukemia cell line, HL-60, we studied the membrane transport of Ara-C before and after differentiation induced by retinoic acid (RA). In RA-treated cells, Ara-C transport was reduced and there was a concomitant increase of the ID50 values of Ara-C in comparison with the controls. By three different procedures to synchronize untreated cells, i.e. density arrest G1 phase enrichment, aphidicolin-induced S phase accumulation and the double isoleucine block method, we found that Ara-C transport was 30-50% higher in the S phase than in the G1 phase. Therefore, the observed decrease in Ara-C transport is, in part, due to the retarded growth accompanied by an accumulation of cells in the G1 phase after differentiation induction.

Topics: Aphidicolin; Biological Transport; Cell Cycle; Cell Differentiation; Cell Membrane; Cytarabine; Deoxycytidine Kinase; Diterpenes; Humans; Interphase; Leukemia, Promyelocytic, Acute; Mutation; Tretinoin; Tumor Cells, Cultured