zinostatin and Neuroblastoma

Neuroblastoma is among the most common malignancies of childhood. Despite greatly improved therapy for some pediatric tumors, the prognosis for children with metastatic neuroblastoma has not changed significantly in the past 10 years. With conventional chemotherapy, radiation therapy, and surgery, children with metastatic neuroblastoma have a 20% long-term survival rate. We describe here approaches to neuroblastoma that target its neuronal characteristics. On the one hand, the neurotransmitter receptors on the surface of the neuroblastoma cells and, on the other hand, specific isozymes that distinguish neuroblastoma cells from their normal counterparts are the focus of these experimental therapies. In the former case, specificity for tumor cells is effected by (1) selective protection of normal neuronal elements from toxicity, or (2) selective potentiation of toxicity for neural tumor cells. It is hoped that these strategies will be generalizable to other neural crest-derived tumors.

Topics: Amifostine; Animals; Dopamine; Drug Synergism; Hydroxydopamines; Mice; Nervous System Neoplasms; Neural Crest; Neuroblastoma; Zinostatin

To identify and mathematically model molecular predictors of response to the enediyne chemotherapeutic agent, neocarzinostatin, in nervous system cancer cell lines.. Human neuroblastoma, breast cancer, glioma, and medulloblastoma cell lines were maintained in culture. Content of caspase-3 and Bcl-2, respectively, was determined relative to actin content for each cell line by Western blotting and optical densitometry. For each cell line, sensitivity to neocarzinostatin was determined. Brain tumor cell lines were stably transfected with human Bcl-2 cDNA cloned into the pcDNA3 plasmid vector.. In human tumor cell lines of different tissue origins, sensitivity to neocarzinostatin is proportional to the product of the relative contents of Bcl-2 and caspase-3 (r (2) = 0.9; P < 0.01). Neuroblastoma and brain tumor cell lines are particularly sensitive to neocarzinostatin; the sensitivity of brain tumor lines to neocarzinostatin is enhanced by transfection with an expression construct for Bcl-2 and is proportional in transfected cells to the product of the relative contents of Bcl-2 and caspase-3 (r (2) = 0.7).. These studies underscore the potential of molecular profiling in identifying effective chemotherapeutic paradigms for cancer in general and tumors of the nervous system in particular.

Topics: Antibiotics, Antineoplastic; Biomarkers, Tumor; Caspase 3; Cell Line, Tumor; Cell Survival; Drug Resistance; Drug Screening Assays, Antitumor; Gene Expression Profiling; Glioma; Humans; Medulloblastoma; Nervous System Neoplasms; Neuroblastoma; Predictive Value of Tests; Proto-Oncogene Proteins c-bcl-2; Zinostatin

Studies of the response of neural crest tumor cells to the DNA cleaving antimitotic agent, neocarzinostatin, have left unanswered the question of whether the DNA cleavage per se or the antimitotic effect is responsible for this response. Furthermore, they do not define the timeframe within which a cell commits to its fate. Using the reversible microtubule-active agent, vinblastine, we now demonstrate that mitotic arrest, even without DNA cleavage, results in the same cellular changes as those seen with neocarzinostatin treatment. The commitment of the cell to its fate occurs within a 15 min treatment with vinblastine, and requires new protein synthesis. The immediate early gene products, c-Fos and c-Jun, appear not to be determinants of this process.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Count; Cell Differentiation; Cells, Cultured; Chromatin; Cycloheximide; Humans; Microtubules; Mitosis; Neuroblastoma; Oncogene Proteins v-fos; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-jun; Vinblastine; Zinostatin

Chemotherapeutic agent-induced DNA cleavage gives rise to apoptosis in a subpopulation of SK-N-SH human neuroblastoma cells; the remaining cells undergo Schwann cell-like differentiation. Like other neural crest and primitive neurectodermal tumor-derived cell lines, SK-N-SH cultures contain cells of neural (N-type) and epithelial (substrate-adherent, or S-type) phenotypes. Using isolated N-type and S-type cells from neuroblastoma, medulloblastoma, melanoma and glioma cell lines, we demonstrate that the determinants of the response to DNA cleavage are intrinsic properties of the cell. Furthermore, using a series of analogues of enediyne deoxyribonucleic acid (DNA) cleaving agents, we show that the molecular target of these agents is likely to be the same in N- and S-type cells, implying that the difference in response characteristics is a function of different distal pathways that are triggered by DNA cleavage. We demonstrate that the concentration of the DNA damaging agent used, and not the specific characteristics of the damage it produces, is the trigger for production of the cellular response. Response type does not correlate with previously published values for expression of the apoptosis modulators Bcl-2, Bcl-XL, wildtype p53, or, in medulloblastoma lines, p75.

Topics: Apoptosis; DNA; DNA Damage; Humans; Neuroblastoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Nerve Growth Factor; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Zinostatin

Prevention by nerve growth factor (NGF) of apoptotic death in neural cells has been variously ascribed to binding of NGF to its low-affinity (p75) or high-affinity (trkA) receptor or to a cooperative interaction between the two. In a series of studies using, in turn, neuroblastoma cell lines that express only p75, mutant NGF species that bind selectively to either p75 or trkA, and a polyclonal antibody that binds to the NGF-binding domain of p75, we demonstrate that NGF binding to p75 is both necessary and sufficient for the abrogation of apoptosis in neuroblastoma cells treated with antimitotic agents.

Topics: Antineoplastic Agents; Apoptosis; Bacterial Proteins; Biomarkers; Cell Adhesion; Humans; Mutagenesis, Site-Directed; Nerve Growth Factors; Neural Crest; Neuroblastoma; Neuroprotective Agents; Receptor, trkA; Receptors, Nerve Growth Factor; Tumor Cells, Cultured; Zinostatin

Neocarzinostatin (NCS) is a naturally occurring enediyne antitumor agent that produces single- and double-strand breaks in cellular DNA. We have previously shown that treatment of human (SK-N-SH) and murine (NB41A3) neuroblastoma cells with NCS results in cell death for a subpopulation within the culture. The remaining cells undergo mitotic arrest with morphological differentiation along glial lines. Further investigation of cell death induced by this agent demonstrates that within 24 hr after a single one hr exposure to submicromolar concentrations of NCS, susceptible cells of both lines decrease in size, round up, detach from the culture surface and fragment in the overlying medium. This cytotoxicity is attenuated by the addition of cycloheximide (in NB41A3 cells) or aurintricarboxylic acid (in NB41A3 and SK-N-SH cells). Fluorescence and electron microscopic examination of the nonadherent cells reveals the chromatin condensation and fragmentation characteristic of apoptosis. Examination of the time course of DNA cleavage reveals that despite the presence of alkaline elution-detectable DNA cleavage, oligonucleosomal-sized DNA fragments are not demonstrable by gel electrophoresis immediately after a 1-hr incubation with the drug (1.6-10,000 nM). However, by 6 hr after treatment, DNA ladders are in evidence at all concentrations of NCS. These results suggest that the oligonucleosomal cleavage of DNA seen after NCS treatment is associated with apoptosis, rather than being the direct result of the strand-cleaving effects of the drug itself.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Death; Cell Nucleus; Cycloheximide; Dactinomycin; DNA Damage; DNA, Neoplasm; Endonucleases; Humans; Mice; Neuroblastoma; Tumor Cells, Cultured; Zinostatin

It has been suggested that G0 and Gi play a role in the collapse of axonal growth cones and the retraction of neurites. We have studied the G-protein content of neuroblastoma cells undergoing neurite outgrowth and subsequent retraction in response to neocarzinostatin (NCS). Stimulators of G0 and Gi have no effect upon neurite outgrowth or retraction and the cellular content of G0 and Gi does not change during the course of these morphological phenomena. Modulation of G-protein content, therefore, most likely does not play a role in this process.

Topics: Animals; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression; GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Neurites; Neuroblastoma; Peptides; Wasp Venoms; Zinostatin

The use of differentiation-inducing agents has been proposed for the purging of bone marrow and for the treatment of minimal residual disease prior to autologous bone marrow transplantation in patients with metastatic neuroblastoma. The present studies examine the effects of the enediyne differentiation inducer neocarzinostatin (NCS) on tumor development from subcutaneous implants of murine (Neuro-2A) neuroblastoma cells. Prior in vitro treatment with NCS results in a concentration- and drug exposure time-dependent decrease in the incidence of tumors from subcutaneously implanted cells. In vivo treatment results in a dose-dependent decrease in the rate of tumor growth. These results imply that enediynes such as NCS may be useful in ex vivo purging regimens and in in vivo treatment of microscopic residual disease in patients with neuroblastoma.

Topics: Animals; Cell Differentiation; Dose-Response Relationship, Drug; Male; Mice; Neoplasm Transplantation; Neuroblastoma; Tumor Cells, Cultured; Zinostatin

Neural crest tumor cells which have been pharmacologically induced in culture to undergo neuronal 'differentiation' have been proposed as a model for normal neural crest cell differentiation. We have previously reported that murine neuroblastoma cells treated with the antineoplastic agent neocarzinostatin (NCS) adopt the light microscopic appearance of differentiated neurons. After undergoing morphologic change, the cells no longer divide. As part of an effort to compare the process of differentiation in these cells with what is known about normal neural crest cells, we have examined the cellular distribution and isoform complement of neural cell adhesion molecules (NCAMs) in native and NCS-treated neuroblastoma cells. Our studies show that NCS induces profound changes in NCAM distribution. Immunohistochemical staining indicates that, in contrast to native neuroblastoma cells, more than 80% of treated cells display surface NCAM by 4 days following treatment. Unlike the case for normal neurons, NCAM is uniformly distributed over the treated cell surface. Neuroblastoma cells treated with NCS are more avidly adherent to culture plates coated with NCAM than are control neuroblastoma cells, reflecting the homophilic binding characteristics of NCAM. Interestingly, Western blot analysis for NCAM demonstrates similar total cellular content of a single NCAM species in both control and treated neuroblastoma cells. Furthermore, this 120 kDa mol. wt. NCAM is an isoform of NCAM not found on normally differentiated cerebellar neurons. While the presence of NCAM on these treated murine neuroblastoma cells is evidence for 'differentiation' along neuronal lines, the isoform complement and cell surface distribution of NCAM in treated cells are not normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Cell Membrane; Cytoplasm; Mice; Molecular Weight; Neuroblastoma; Neurons; Tumor Cells, Cultured; Zinostatin

Neocarzinostatin is an antineoplastic agent that induces differentiated morphology in human (SK-N-SH) neuroblastoma cells in culture. We have compared this morphological differentiation with that induced by the endogenous differentiation inducer, nerve growth factor (NGF), and have explored the effects of exposure to NGF upon the morphological changes induced by neocarzinostatin in SK-N-SH cells. Both NGF and neocarzinostatin induced process outgrowth in these cells. The processes formed in the presence of NGF however, were shorter and thinner than those induced by neocarzinostatin. Furthermore, only neocarzinostatin induced enlargement of the somata of the cells, and caused cell death in a concentration-dependent fraction of the culture. These distinguishing features of treated cells allowed us to determine whether or not NGF exposure altered responsiveness of the cells to neocarzinostatin. NGF (100-1000 ng/mL) protected SK-N-SH cells from the morphological and cytocidal effects of neocarzinostatin (1-hr exposure, 0.017 to 0.033 micrograms/mL). Protection from neocarzinostatin required that NGF be continuously present for a period beginning 24 hr prior to neocarzinostatin exposure and continuing for the duration of the experiment, implying that the protection afforded by NGF has a latency necessitating pretreatment, and is reversible. These results suggest that neocarzinostatin is taken up by the cells and can exert its effects once NGF is removed, even after neocarzinostatin is washed out of the medium. The signal transduction cascade triggered by NGF receptor binding may prevent the action of neocarzinostatin or the expression of the cellular changes induced in SK-N-SH cells by neocarzinostatin.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Survival; Drug Interactions; Humans; Mice; Nerve Growth Factors; Neuroblastoma; Tumor Cells, Cultured; Zinostatin

Neuroblastoma is among the most common malignancies of childhood. Despite greatly improved therapy for some pediatric tumors, the prognosis for children with metastatic neuroblastoma has not changed significantly in the past 10 years. With conventional chemotherapy, radiation therapy, and surgery, children with metastatic neuroblastoma have a 20% long-term survival rate. We have pursued novel chemotherapeutic approaches to neuroblastoma that target the neurotransmitter receptors on the surface of these cells. Specificity for these neural crest tumor cells is effected by (1) selective protection of normal neuronal elements from toxicity, or (2) selective potentiation of toxicity for neural tumor cells. In the first instance, the oxygen radical-generating neurotransmitter analogue 6-hydroxydopamine is used as a neural crest-specific toxin. Normal neural crest cells are protected from this toxicity by oxygen radical-scavenging analogues of the compound WR2721, which is specifically taken up by nonneoplastic cells. In the second instance, neocarzinostatin, an antineoplastic natural product that must be activated by thiol groups to be toxic, is used in conjunction with 6-mercaptodopamine, a thiol-containing compound that gains specific entry into neural crest cells by virtue of its neurotransmitter-like structure. We have found that neocarzinostatin induces morphologic differentiation of neuroblastoma cells, and we are also currently characterizing the biochemical accompaniments of this morphologic change.

Topics: Adolescent; Amifostine; Animals; Antineoplastic Agents; Drug Synergism; Humans; Infant; Infant, Newborn; Mice; Neural Crest; Neuroblastoma; Oxidopamine; Sulfhydryl Compounds; Tumor Cells, Cultured; Zinostatin

The development of chemotherapeutic approaches to cancer has been hampered by the toxicity of proposed agents for normal rapidly dividing cells. By using neocarzinostatin, a "pro-drug" which is activated by reduction by thiol compounds, adjunctively with 6-mercaptodopamine, a thiol-containing dopamine analogue, we have been able to enhance neocarzinostatin toxicity for cells of the neural crest tumor neuroblastoma. Thiol compounds that are not neurotransmitter analogues do not act synergistically with neocarzinostatin in this system. Since most normal rapidly dividing cells do not have surface dopamine receptors, we propose this approach for the selective targeting of toxicity for neuroblastoma cells. We further introduce cell-selective activation of prodrugs as a new chemotherapeutic strategy which demands further development.

Topics: Animals; Dopamine; Drug Synergism; Mice; Neuroblastoma; Sulfhydryl Compounds; Tumor Cells, Cultured; Zinostatin

Neocarzinostatin, an antineoplastic agent which consists of a chromophore noncovalently bound to a protein of approximately 10,000D MW, induces the morphological differentiation of cells of the C1300 murine neuroblastoma line in vitro. It also has antimitotic activity, ascribed previously in other cell lines to the action of the chromophore upon DNA. The chromophore is antimitotic for neuroblastoma cells, as well. The morphology-altering effect of neocarzinostatin can also be mimicked by chromophore alone. The morphology-altering effect becomes apparent at doses of the agent which decrease the culture growth rate to approximately 50% of control values. It appears that induction of morphological neural differentiation of neuroblastoma cells requires an increase in the doubling time above a critical value.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; Mice; Neoplasm Proteins; Neuroblastoma; Neurons; Tumor Cells, Cultured; Zinostatin