aphidicolin and Leukemia

Although the mechanism of action responsible for the effects of low-dose ara-C remains unclear, certain insights are available concerning the interaction of this agent with DNA. Ara-C incorporates into DNA, and the extent of (ara-C)DNA formation correlates significantly with loss of clonogenic survival. The inhibition of DNA replication by ara-C also results in DNA fragmentation and terminal differentiation of leukemic cells. Other studies have demonstrated that inhibition of DNA replication by ara-C results in an aberrant form of DNA synthesis with certain segments of DNA being replicated more than once within a single cell cycle. The additional copies of certain segments of DNA could lead to the accumulation of DNA fragments and alterations in gene expression. It is of interest that other inhibitors of S-phase DNA replication such as aphidicolin and hydroxyurea can also induce similar phenotypic changes in HL-60 and K562 leukemia cells. Although the in vitro data support the concept that ara-C is capable of inducing leukemic cell differentiation, there is no evidence to suggest that this agent induces differentiation of human leukemic cells in vivo. Drug levels achieved by administration of low-dose ara-C (42-64 nmol/L) result in the incorporation of ara-C into bone marrow mononuclear preparations from patients with preleukemia syndromes. This concentration of ara-C (5 X 10(-8) mol/L) slows DNA replication of human leukemic cells in vitro. Thus, the clinical use of low doses of ara-C that achieve plasma concentrations of 10(-8) to 10(-7) mol/L could theoretically induce maturational effects by partially inhibiting DNA synthesis. At the present time we have no available data to support this contention. On the basis of chromosomal analyses, low-dose ara-C apparently maintains sufficient drug levels to suppress more "malignant" clones, but even "clonal" selection may represent elimination of leukemic cells by either cytotoxicity or induction of terminal differentiation. Further studies will be necessary to define the mechanism of action of low-dose ara-C in preleukemia.

Topics: Adult; Aged; Aphidicolin; Bone Marrow; Cell Differentiation; Cell Division; Cell Line; Cytarabine; Diterpenes; DNA; DNA Replication; Dose-Response Relationship, Drug; Female; Hemoglobins; Humans; Karyotyping; Kinetics; Leukemia; Male; Middle Aged; Preleukemia

The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested as a novel molecular target-based therapy for acute myeloid leukemia. Several studies have established the role of ERK in cell cycle progression from G(1) to S phase in response to mitogen, but the role of ERK after the restriction point is less clarified. In this study, we used models of aphidicolin and nocodazole-synchronized HL-60 and NB4 leukemia cell lines to determine the kinetics of ERK activity during the progression of the cell cycle and to test the effects of commercially available inhibitors on G(2)/M progression of synchronized leukemia cells. In aphidicolin-synchronized cells, the activity of ERK was low during early S phase and increased at late S and G(2)/M phase of the cell cycle. The presence of MEK inhibitors PD 98059 and U0126 caused a delay in G(2)/M phase. In nocodazole-synchronized cells, the activity of ERK was low during M/G(1) transition and MEK inhibitors had no effects on return of the cells to G(1) phase. These results demonstrate that the activity of ERK is required during G(2)/M phase of leukemia cell cycle before the cells reach metaphase-anaphase transition.

Topics: Anaphase; Aphidicolin; Cell Cycle; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; G2 Phase; Humans; Leukemia; Metaphase; Nocodazole

Influx of Ca(2+) represents an important regulatory signal in the process of cell proliferation. However, little is known about how Ca(2+) entry changes during the cell-cycle. Patch-clamp experiments and microfluorimetry show that store-operated Ca(2+) entry was substantially reduced in rat basophilic leukaemia cells cultured for 24h under serum-free conditions. Likewise, retinoic acid treatment blocked Ca(2+) influx activated by store depletion via inositol 1,4,5-trisphosphate. Both procedures are known to arrest cells at the G0/G1 boundary of the cell-cycle and induced a reduction in 5-bromo 2'-deoxyuridine incorporation into DNA. Ca(2+) release from the stores remained unaltered and two types of K(+) currents were not affected in cells after serum starvation. The specific reduction in Ca(2+) entry was not detected when using aphidicolin, 5-fluorouracil or thymidine to synchronise the cell-cycle. These data suggest that store-operated Ca(2+) influx changed during cell-cycle progression which might have important implications for cell growth.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Aphidicolin; Bromodeoxyuridine; Calcium; Cell Cycle; Cell Division; Culture Media, Serum-Free; Enzyme Inhibitors; Fluorouracil; Guanosine 5'-O-(3-Thiotriphosphate); Inositol 1,4,5-Trisphosphate; Leukemia; Patch-Clamp Techniques; Potassium; Potassium Channels; Rats; Time Factors; Tretinoin; Tumor Cells, Cultured

The growth-suppressive activity of the retinoblastoma (RB) protein is suggested to be regulated by phosphorylation. In studies on the kinase that phosphorylates the RB proteins, we have previously found that RB proteins can be phosphorylated by purified cdc2 kinase. In this study, we noted that RB proteins immunoprecipitated from human cell lysates are weakly phosphorylated in the absence of purified cdc2 kinase. Immunoblot analysis showed the presence of p34cdc2 in the immunoprecipitates with anti-RB monoclonal antibody. In addition, the coprecipitated kinase was found to have the same substrate specificity as cdc2 kinase. The associated kinase activity was particularly high in cells arrested in G1/S and S phase by aphidicolin. Furthermore, RB proteins were shown to be phosphorylated in nuclear extracts by some endogenous cdc2-like kinase(s). These results suggest that cdc2-like kinase is the main kinase for phosphorylation of RB proteins in vivo.

Topics: Amino Acid Sequence; Animals; Aphidicolin; CDC2 Protein Kinase; Cell Cycle; Cell Line, Transformed; Cell Nucleus; Colonic Neoplasms; HeLa Cells; Humans; Leukemia; Mammary Neoplasms, Experimental; Mice; Molecular Sequence Data; Neuroblastoma; Peptides; Phosphorylation; Retinoblastoma Protein; Substrate Specificity

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Topics: Actin Cytoskeleton; Aphidicolin; Camptothecin; Cell Death; Cell Line; Cell Survival; Cytochalasin B; Humans; Ionomycin; Kinetics; Leukemia; Tetradecanoylphorbol Acetate; Ultraviolet Rays

The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.

Topics: Aphidicolin; Arabinofuranosylcytosine Triphosphate; Deoxyguanine Nucleotides; Diterpenes; DNA Primase; DNA-Directed DNA Polymerase; DNA, Neoplasm; Humans; Leukemia; RNA; RNA Nucleotidyltransferases; Tumor Cells, Cultured; Vidarabine Phosphate

In continuing search for exploitable biochemical differences between cancer and normal cells at the level of DNA replication, leukemic and "normal" hematopoietic cells from four different, established human cell lines were grown in culture flasks, and both the DNA and the DNA polymerase alpha were isolated in each case from the harvested (5-10 g wet weight) cell pellets. The four selected cell lines included a "normal" lymphoblastoid B-cell line (RPMI-1788), a pre-B cell (NALM-6) and a T-cell (MOLT-4) acute lymphoblastic leukemias, and a promyelocytic leukemia (HL-60). The DNA polymerase alpha enzyme of the two B-cell lines (both the leukemic and the "normal") showed the usual sensitivity toward inhibition by aphidicolin, while those from the two other leukemic cell lines were remarkably resistant to the antibiotic. Partially thiolated polycytidylic acid (MPC) strongly inhibited only the DNA polymerase alpha of the "normal" cell line, whereas the corresponding enzymes of all three leukemic cell lines were relatively insensitive to MPC. In contrast, the partially thiolated DNAs derived from the leukemic cell lines more strongly inhibited the DNA polymerase alphas of the leukemic cell lines than that of the "normal" cell line. These results indicate the existence of some structural differences between the DNA polymerase alpha enzymes (as well as between the DNAs) of human cells of different lineage and, particularly, of leukemic vs. "normal" character; such differences could be exploited in the design of selective antitemplates for chemotherapy.

Topics: Animals; Aphidicolin; Cattle; Cell Line; Diterpenes; DNA; DNA Polymerase II; Ethylmaleimide; Humans; Leukemia; Magnesium; Manganese; Sulfhydryl Compounds

Aphidicolin inhibits DNA replication and growth of all tested human and murine neoplastic cells including leukemic T- and B-lymphocytes and melanocarcinoma cells. The concentration of aphidicolin causing 50% inhibition of DNA synthesis in all of the tested neoplastic cell lines is similar to that necessary to inhibit DNA synthesis in HeLa cells by 50%. The mechanism of inhibition of DNA synthesis in neoplastic cells is again due to the inhibition of DNA polymerase alpha by aphidicolin. Aphidicolin at a concentration 100 times higher than that causing 50% inhibition of DNA synthesis and cell growth had no effect on total protein synthesis, on the secretion of immunoglobulins, or on the expression of HLA antigens which are involved in relevant phenomena of the immune response.

Topics: Animals; Aphidicolin; Cell Division; Cell Line; Diterpenes; DNA Polymerase II; DNA Replication; HLA Antigens; Humans; Immunoglobulins; Leukemia; Lymphocytes; Melanoma; Mice; Multiple Myeloma; Protein Biosynthesis