Determinants of the response of neuroblastoma cells to DNA damage: the roles of pre-treatment cell morphology and chemical nature of the damage.

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.

Induction of apoptosis in murine and human neuroblastoma cell lines by the enediyne natural product neocarzinostatin.

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.

Bay K 8644 is a negative inotrope in the presence of arachidonic acid but not eicosapentaenoic acid.

The inotropic and radioligand binding characteristics of arachidonic acid (AA) and the fish oil derivative, eicosapentaenoic acid (EPA), were studied. AA alone was a concentration-dependent negative inotrope in isolated hamster papillary muscles. The negative inotropic effect of AA was enhanced 500-fold by the coadministration of indomethacin (cyclo-oxygenase inhibitor) and caffeic acid (lipoxygenase inhibitor). The dihydropyridine calcium channel agonist Bay K 8644 (BK) was a concentration-dependent positive inotrope. BK in the presence of AA was a concentration-dependent negative inotrope. The negative inotropic effect of BK in the presence of AA was blocked by caffeic acid but not indomethacin. EPA alone was a concentration-dependent negative inotrope. EPA had no effect on the positive inotropic effect of BK. Scatchard analyses of [3H]nitrendipine (dihydropyridine) binding to cardiac membranes in the presence of AA or EPA resulted in a decrease in the KD (-1/slope) with no effect on Bmax (x intercept). Kinetic studies further revealed that both AA and EPA enhanced the off rate of [3H]nitrendipine binding. These data suggest that both AA and EPA mediate similar direct negative inotropic effects through cardiac sarcolemmal calcium channels. A unique effect on E-C coupling by AA, not shared by EPA, is mediated through the lipoxygenase pathway (leukotrienes). AA and its eicosanoid products may play critically important roles in regulating myocardial contractility during acute and chronic myocardial ischemia. Differences between AA and EPA may contribute to the beneficial effects of dietary fish oils enriched in EPA.

Regulation of [3H]nitrendipine binding by phospholipases A2 and C through direct and GTP-sensitive mechanisms.

We compared the effects of Phospholipases A2, C, B and D on [3H]nitrendipine binding to hamster cardiac membranes, in the absence and presence of ATP or GTP. Phospholipase A2, competitively inhibited [3H]nitrendipine binding to hamster cardiac membranes unchanged by ATP or GTP (Ki = 5 ng/ml); as evidenced by complete and reversible displacement of [3H]nitrendipine binding and increase in KD on Scatchard analyses. Phospholipase C also completely inhibited [3H]nitrendipine binding to hamster cardiac membranes (Ki = 5 micrograms/ml) with a decrease in Bmax and no change in KD on Scatchard analyses. The addition of GTP alone inhibited the PLC effect in EGTA-treated membranes. The addition of GTP with either CaCl2 or ATP or both resulted in an equal and opposite enhancement of the PLC effect. Phospholipases B and D had no effect on [3H]nitrendipine binding. These data support: (1) Direct effect of PLA2 on dihydropyridine binding. (2) Indirect regulation of dihydropyridine binding by Phospholipase C through a GTP and ATP-sensitive mechanism.