aphidicolin and Neuroblastoma

Aphidicolin, a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, is under investigation as anti-cancer drug. Because of its poor solubility in water, it cannot be administered directly in vivo. Systemic application of aphidicolin glycinate or aphidicolin gamma-cyclodextrin complexes resulted in tumour growth inhibition but not in cures. To improve the pharmacokinetics, a liposomal preparation of aphidicolin was developed and tested in neuroblastoma-bearing (UKF-NB-3) mice. The loading capacity of these liposomes was limited. Therefore, 4.5 mg aphidicolin/kg body weight was the maximum aphidicolin dose that could be applied as liposomal preparation in this approach. Comparison of effects on tumour growth exhibited by aphidicolin liposomes (4.5 mg aphidicolin/kg) given for 15 consecutive days to those of gamma-cyclodextrin inclusion complexes (15 mg aphidicolin/kg) revealed comparable tumour growth inhibition, although aphidicolin concentrations were approximately 3-fold lower. This shows that liposomal encapsulation is a promising strategy for the improvement of systemic anti-cancer activity of aphidicolin.

Topics: Animals; Antibiotics, Antineoplastic; Aphidicolin; Drug Carriers; Female; Humans; Liposomes; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Tumor Cells, Cultured

The expression of folate sensitive and aphidicolin induced fragile sites in the blood lymphocyte chromosomes of affected and unaffected members from 2 neuroblastoma families were studied. The subjects included 4 neuroblastoma patients, and 9 of their clinically healthy first degree relatives and corresponding number of age and sex matched controls. Lymphocytes cultured in folate deprived culture medium showed rare fragile sites at band p13.1 of chromosome 1, in a frequency of 3%-5% in all the 4 neuroblastoma patients. In aphidicolin treated cultures, the patients and unaffected members in neuroblastoma families, showed hypersensitivity to aphidicolin, as evidenced by the significant increase in percentage of aberration/cell (ab/c) and damaged cells (dc), over that of controls (P < 0.01). Aphidicolin induced fragile sites were more pronounced in chromosomes 1 and 2. A larger number of subjects have to be studied to prove whether altered fragile site expression may be a cytogenetic evidence for an individual or familial cancer predisposing genetic constitution.

Topics: Aphidicolin; Case-Control Studies; Cells, Cultured; Child, Preschool; Chromosome Aberrations; Chromosome Banding; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 2; Enzyme Inhibitors; Female; Folic Acid; Genetic Predisposition to Disease; Hematinics; Humans; Infant; Karyotyping; Lymphocytes; Male; Neuroblastoma; Pedigree

Neuroblastoma is the second most common solid malignancy of childhood. Enhanced expression of the amplified N-myc gene in the tumor cells may be associated with poor patient prognosis and may contribute to tumor development and progression. The use of deferoxamine mesylate (DFO), an iron chelator, to treat neuroblastoma is being investigated in national clinical studies. We show here by TUNEL assay and DNA laddering that DFO induces apoptosis in cultured human neuroblastoma cells, which is preceded by a decrease in the expression of N-myc and the altered expression of some other oncogenes (up-regulating c-fos and down-regulating c-myb) but not housekeeping genes. The decrease in N-myc expression is iron-specific but does not result from inhibition of ribonucleotide reductase, because specific inhibition of this iron-containing enzyme by hydroxyurea does not affect N-myc protein levels. Nuclear run-on and transient reporter gene expression experiments show that the decrease in N-myc expression occurs at the level of initiation of transcription and by inhibiting N-myc promoter activity. Comparison across neuroblastoma cell lines of the amount of residual cellular N-myc protein with the extent of apoptosis measured as pan-caspase activity after 48 h of iron chelation reveals no correlation, suggesting that the decrease in N-myc expression is unlikely to mediate apoptosis. In conclusion, chelation of cellular iron by DFO may alter the expression of multiple genes affecting the malignant phenotype by multiple pathways. Given the clinical importance of N-myc overexpression in neuroblastoma malignancy, decreasing N-myc expression by DFO might be useful as an adjunct to current

Topics: Aphidicolin; Apoptosis; Deferoxamine; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Reporter; Humans; Hydroxyurea; Inhibitory Concentration 50; Iron; Iron Chelating Agents; Neuroblastoma; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; RNA, Messenger; Substrate Specificity; Transcription, Genetic; Tumor Cells, Cultured

Treatment failure in most neuroblastoma (NB) patients is related to primary and/or acquired resistance to conventional chemotherapeutic agents. Aphidicolin (APH), a tetracyclic diterpene, exhibits specific cytotoxic action against NB cells. The purpose of this study was to compare antitumoral efficacy of APH in parental NB cell lines and cell subclones that exhibit drug resistance to vincristine (VCR), doxorubicin (DOX) and cisplatin. Due to poor solubility of APH in water, gamma-cyclodextrin (gamma-CD) inclusion complexes of APH were used for systemic treatment of xenotransplanted parental and VCR-resistant UKF-NB-3 tumours. APH and its gamma-CD inclusion complexes inhibited growth of parental and drug-resistant NB cells at equimolar doses in vitro. Growth of VCR-sensitive and -resistant NB tumors was inhibited at equal doses in a dose-dependent fashion in vivo. These results indicate that the specific cytotoxic activity of APH against NB cells in vitro and in vivo is independent of cellular mechanisms facilitating drug resistance to conventional chemotherapeutic drugs. Hence, taking into account our previous findings that APH acts synergistically with VCR and DOX, APH might be an additive tool for the therapy of NB and is suitable for evaluation in clinical studies of NB treatment protocols.

Topics: Animals; Antineoplastic Agents; Aphidicolin; Body Weight; Cell Survival; Cyclodextrins; Drug Carriers; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Humans; Mice; Mice, Nude; Neuroblastoma; Tumor Cells, Cultured

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell's proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA alpha-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell's proliferative status.

Topics: Acetylcholine; Animals; Aphidicolin; Carrier Proteins; Cell Differentiation; Cell Division; Choline; Choline O-Acetyltransferase; DNA Polymerase III; Drug Synergism; Enzyme Inhibitors; Membrane Transport Proteins; Mice; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Disseminated neuroblastoma diseases are still indicated by a poor outcome despite treatment regimens including radiation therapy and high-dose chemotherapy with stem cell rescue. Therefore, new substances and treatment regimens are of interest. Aphidicolin (APH), a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, has a specific toxicity for neuroblastoma cells. Furthermore, it was shown to enhance the effects of X-ray radiation and chemotherapy on malignant cells. To find new substances, 20 APH derivatives were tested for their anti-neuroblastoma efficacy in vitro in UKF-NB-2 cells. Five derivatives had antitumoral activity in neuroblastoma cells. A relationship between the structure and the antitumoral efficacy showed that the hydroxyl groups at C-3 and C-18 are essential for the antitumoral effects. Furthermore, antitumoral effects of APH in combination with doxorubicin and vincristine, both part of commonly used treatment regimens for disseminated neuroblastoma diseases, were tested in the neuroblastoma cell line UKF-NB-2. APH was found to act synergistically with vincristine and synergistically to additive with doxorubicin depending on the molecular ratio of the substances in combination. This may offer the chance to use APH and its derivatives as additional tools in the treatment of neuroblastomas.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Aphidicolin; Cell Survival; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Humans; Molecular Structure; Neuroblastoma; Nucleic Acid Synthesis Inhibitors; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vincristine

Aphidicolin is a tetracyclic diterpene antibiotic which kills human neuroblastoma cells (NB) in vitro while it has no significant effect on the viability of different human cell types including normal embryonal cells. In the present study, we tested whether aphidicolin encapsulated in liposomes kills NB cells with the efficacy superior to that of unencapsulated aphidicolin. The drug was entrapped in vesicles composed of phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of 83:5:12. The treatment with encapsulated aphidicolin at a concentration of 200 nmol for 5 days killed all cells of three human NB cell lines. In contrast, at least 30% of the cells survived 5 days of treatment with 200 nmol unencapsulated aphidicolin. The results showed that aphidicolin killing of human NB cells may be increased by encapsulation in liposomes.

Topics: Aphidicolin; Cell Death; DNA Polymerase II; Drug Carriers; Enzyme Inhibitors; Humans; Liposomes; Neuroblastoma; Tumor Cells, Cultured

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

Topics: Aphidicolin; Base Sequence; Blotting, Western; Cell Differentiation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Humans; Molecular Sequence Data; Nerve Growth Factors; Neuroblastoma; Neurons; Oligonucleotides, Antisense; Precipitin Tests; Tumor Cells, Cultured

We examined events associated with neuronal differentiation of neuroblastoma cell line SH-SY5Y. Treatment with nerve growth factor (NGF) and aphidicolin, an inhibitor of DNA polymerases alpha and delta, induces terminal differentiation of SH-SY5Y cells. Following 3-4 days of treatment with NGF + aphidicolin, the cells irreversibly ceased proliferation and differentiated. There was a succession of events preceding differentiation. Down-regulation of c-myc was an early event occurring after less than 1 day of treatment with NGF + aphidicolin. Upregulation of the trkA and low-affinity NGF receptors (LNGFR) occurred after 3 days of NGF + aphidicolin treatment and required treatment with both NGF and aphidicolin. To test the role of TrkA in neuroblastic differentiation, we transfected SH-SY5Y cells with a TrkA-expression plasmid. In response to NGF in the absence of aphidicolin, the TrkA-transformant line ceased proliferation and irreversibly differentiated. SH-SY5Y cells bearing a control plasmid displayed only modest, reversible differentiation and did not cease cell proliferation in response to NGF. Hence, expression of NGF receptors is upregulated during differentiation of SH-SY5Y cells, and overexpression of TrkA enhances NGF-induced differentiation.

Topics: Aphidicolin; Cell Differentiation; Gene Expression Regulation; Nerve Growth Factors; Neuroblastoma; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptors, Nerve Growth Factor; Tumor Cells, Cultured

Iron (Fe) is known to be necessary for cellular proliferation. Previous studies have suggested that neuroblastoma cells appear to be relatively sensitive to growth inhibition by a specific Fe chelator, deferrioxamine (DFO), in vitro. Also, DFO has been recently used for the treatment of neuroblastoma patients. In this paper we demonstrate that neuroblastoma cell proliferation in vitro is extremely sensitive to inhibition by DFO as compared to another cell line with almost identical growth kinetics. Neuroblastoma cells treated with DFO adapt appropriately to Fe chelation as measured by marked upregulation of transferrin receptor mRNA, increased functional transferrin receptor, and decreased cellular ferritin concentration. Further studies that quantitated cellular incorporation of 59Fe from added transferrin-59Fe in the presence of DFO indicated that neuroblastoma cells were more sensitive to inhibition of Fe incorporation by the chelator as compared to the other cell line. Neuroblastoma cells treated with DFO showed a consistent arrest in the G1 phase of the cell cycle. For cells taken from the "resting" state this block occurred before the vast majority of cells had entered S or G2-M phases of the cell cycle. Further evidence that neuroblastoma cells were arrested before the G1-S interface was provided when cells inhibited by DFO and released into aphidicolin exhibit arrest at the G1-S interface, whereas release from aphidicolin into DFO resulted in entry into S phase. Also, DFO-treated cells exhibited a decrease in both p34cdc2 immunoreactive protein as well as kinase activity. The results of these latter studies strongly indicate evidence for a Fe requirement for malignant cell proliferation before the onset of DNA synthesis. Our results also provide a basis for further studies that will better define a therapeutic approach to patients with neuroblastoma utilizing DFO treatment.

Topics: Aphidicolin; Cell Count; Cell Division; Deferoxamine; Drug Screening Assays, Antitumor; Ferritins; G1 Phase; Glioma; Humans; Iron; Neuroblastoma; Receptors, Transferrin; S Phase; Transferrin; Tumor Cells, Cultured

Aphidicolin is a tetracyclic diterpene antibiotic which is known to inhibit the growth of eucaryotic cells by reversible binding to DNA polymerase alpha without significant effect on cell viability in most common human cell lines. We observed that aphidicolin at a concentration of 5 x 10(-7) M kills all cells of four human neuroblastoma cell lines. In contrast, viability of normal human embryonal cells and of human continuous cell lines including HeLa, H9, A549 and Caco-2 was influenced only moderately by aphidicolin. In addition, neuroblastoma cells were killed after treatment with 5 x 10(-7) M aphidicolin in cocultures with normal embryonal cells which continued to proliferate after removal of aphidicolin. These results show that aphidicolin provides an agent which selectively kills neuroblastoma cells in vitro.

Topics: Antineoplastic Agents; Aphidicolin; Cell Division; Cell Line; Cell Survival; DNA; Drug Screening Assays, Antitumor; Embryo, Mammalian; Humans; Neuroblastoma; 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

The purpose of the present study was to examine the distribution pattern of acridine orange (AO) chromatin interaction products (AOCI) in human neuroblastoma IMR-32 cells and to test whether AO labeling is correlated with BrdU incorporation, and immunohistochemical localization of DNA polymerase alpha, and human N-myc-gene product. Effects of aphidicolin, alpha-amanitin, and actinomycin D on visualization of AO binding to euchromatin and on N-myc-gene expression were also examined. About 25% of the cell nuclei in logarithmic growth phase were immunohistochemically demonstrated to be labeled with BrdU after incubation at 37 degrees for 30 min, indicating cells in DNA synthesis. Most of the cell nuclei showed positive immunoreactivity to DNA polymerase alpha, while human N-myc gene product was found in about 60-80% of the cell nuclei. Electron microscopic studies revealed that about 25% of neuroblastoma cells showed characteristic AOCI within cell nuclei. In the presence of aphidicolin, alpha-amanitin, and actinomycin D, positive cells for N-myc gene product decreased markedly. Percentages of AO positive cells and numbers of AOCI per cell nucleus also showed a marked decrease. But northern blot analysis demonstrated that the expression level of N-myc gene was only repressed by the transcriptional inhibitors alpha-amanitin and actinomycin D. However, no repression was caused by aphidicolin. The present and previous studies of the authors suggest that the ultracytochemical AO method may be indicative for conformational changes of chromatin of cells confined to the cell cycle. Inhibitors of RNA and DNA synthesis then may change the conformational state of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acridine Orange; Amanitins; Aphidicolin; Blotting, Northern; Bromodeoxyuridine; Cell Nucleus; Chromatin; Dactinomycin; Diploidy; Diterpenes; DNA Polymerase II; DNA, Neoplasm; Euchromatin; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Immunohistochemistry; Microscopy, Electron; Neuroblastoma; Proto-Oncogene Proteins c-myc; Tumor Cells, Cultured

We recently demonstrated an increased expression of fragile sites, induced by aphidicolin, in lymphocytes of neuroblastoma patients. We have now extended our studies to parents of affected children with neuroblastoma to verify if this characteristic may be genetically transmitted. We have examined 20 families. In most of them, the hypersensitivity to aphidicolin was found in the affected child and in at least one parent. Moreover, some of the parents showed an increase in the expression of the fragile sites 1p32, 1p13, or both that are preferentially expressed in neuroblastoma patients. The possible relations between the hypersensitivity to aphidicolin and the inheritance of predisposition to neuroblastoma must be clarified.

Topics: Adolescent; Adult; Antibiotics, Antineoplastic; Aphidicolin; Cells, Cultured; Child; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 1; Diterpenes; Drug Hypersensitivity; Female; Humans; Infant; Infant, Newborn; Lymphocytes; Male; Neuroblastoma

To clarify the possible relationship between fragile site expression and cancer, we examined lymphocytes from patients affected by neuroblastoma. This neoplasia may be inherited in some cases and is often characterized by a specific chromosomal aberration: deletion of the short arm of chromosome 1. We found a higher expression of fragile sites after aphidicolin and, to a lesser extent, after methotrexate treatment in lymphocytes from neuroblastoma patients as compared with those of normal donors. The analysis of fragile site distribution pointed out the increase in the expression of fragile site 1p32 in the patients. We believe that this finding might be relevant because this fragile site is located in the same region where breakpoints and rearrangements frequently occur in neuroblastoma cells.

Topics: Adolescent; Adult; Aphidicolin; Bone Marrow; Cells, Cultured; Child; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 1; Diterpenes; Female; Humans; Lymphocytes; Male; Methotrexate; Neuroblastoma

Human SY5Y neuroblastoma cells which were differentiated in culture by treatment with 7S murine nerve growth factor for 5 weeks and selection with aphidicolin (L. Jensen, Dev. Biol. 120:56-64, 1987) demonstrated a considerably slower rate of removal of DNA adducts of benzo[a]pyrene, benzo[a]pyrenediolepoxide, and N7-methylguanine than did undifferentiated mitotic cells. A dramatic decline in unscheduled DNA synthesis induced by UV radiation was similarly observed. DNA polymerase beta and uracil DNA glycosylase were unchanged after differentiation, DNA polymerase alpha and DNA methylase decreased roughly threefold, and total apurinic-apyrimidinic endonuclease activity increased roughly threefold after treatment.

Topics: Aphidicolin; Cell Differentiation; Cell Line; Diterpenes; DNA; DNA Repair; DNA Replication; DNA, Neoplasm; Dose-Response Relationship, Radiation; Humans; Kinetics; Nerve Growth Factors; Neuroblastoma; Ultraviolet Rays

Human neuroblastoma SH-SY5Y (SY5Y) cultures, exposed to murine 7 S nerve growth factor (NGF) for 5 weeks and selected with aphidicolin (Aph) for 1 week, acquire several properties indicative of mature peripheral nerve cells. The mitotic activity of treated cultures decreases prior to Aph selection and ultimately reaches a level approximately 3% that of untreated cultures by Week 4 of treatment. The measured plasma membrane resting potential of the cells increases from -5 mV for untreated cells to -(45-56) mV for NGF/Aph-treated cells. Intracellular stores of monoamines are increased as determined by histochemical staining, and levels of neuron-specific enolase antigen increase as a result of NGF/Aph treatment. The resulting outgrowth of neurites is extensive and large bundles of processes commonly exceed 300 micron in length. NGF/Aph-treated cells acquire a dependence upon NGF for survival; however, with continued administration of NGF, the cultures appear to be capable of surviving indefinitely. Retinoic acid will also promote certain aspects of a differentiated phenotype under similar culture conditions. As judged by these criteria, cells of the SY5Y human neuroblastoma cell line have the potential for phenotypic and irreversible differentiation in vitro and can survive for prolonged periods under these culture conditions.

Topics: Aphidicolin; Cell Differentiation; Cell Line; Cell Membrane; Cell Separation; Cell Survival; Diterpenes; Flow Cytometry; Histocytochemistry; Humans; Membrane Potentials; Microscopy, Electron, Scanning; Mitosis; Nerve Growth Factors; Neuroblastoma; Phosphopyruvate Hydratase; Staining and Labeling

SY5Y neuroblastoma cells were treated with retinoic acid to induce differentiation of neuroblast-like cells and with aphidicolin (an inhibitor of DNA polymerase) to eliminate the flat cells present in the long-term cultures and masking some of the biochemical developmental changes. Catecholaminergic enzyme (tyrosine hydroxylase and monoamine oxidase-A) activity was consistently increased with development and the increase was significantly greater after aphidicolin-induced elimination of dividing, non-neuronal cells.

Topics: Animals; Aphidicolin; Cell Differentiation; Cell Line; Clone Cells; Diterpenes; Isoenzymes; Models, Neurological; Monoamine Oxidase; Neuroblastoma; Neurons; Tretinoin; Tyrosine 3-Monooxygenase