Claudia A. Lipschultz

The influence of Cremophor EL on the cell cycle effects of paclitaxel (Taxol®) in human tumor cell lines

We have perfomed DNA flow analysis, mitotic index studies, time-lapse photography, and paclitaxel uptake studies of human tumor cell lines exposed to paclitaxel. DNA flow analysis demonstrated that cells began accumulating in G2/M within 6 hrs of exposure to paclitaxel; by 12 hrs over 50% of cells accumulated in G2/M at all concentrations tested. After 24 hrs of exposure to 10 nM paclitaxel, cells underwent non-uniform mitotic division resulting in multinucleated cells. Of cells treated with 30 nM to 1000 nM paclitaxel, 75% to 85% remained blocked in G2/M for up to 72 hrs. Although a large proportion of cells treated with higher concentrations of paclitaxel (10,000 nM) was blocked in G2/M, a significant proportion (10% to 40%) of these cells was also in Gl. Cells exposed to lower concentrations of paclitaxel (10 nM to 1000 nM) in medium containing 0.135% (v/v) Cremophor EL also had a relatively large proportion in Gl. Mitotic index studies demonstrated that the paclitaxel-induced G2/M block was initially a mitotic block and that cells remained in mitosis for up to 24 hrs. With additional time of exposure to paclitaxel, mitotic index and time-lapse studies indicated that cells attempted to complete mitosis; however, cytokinesis was inhibited and cells became multinucleated. Time-lapse photography revealed that paclitaxel markedly prolonged the time in mitosis from 0.5 hr to 15 hr. High levels of Cremophor EL (0.135% v/v) markedly reduced the number of cells in mitosis but did not alter the mitotic delay induced by paclitaxel.3H-paclitaxel uptake studies revealed that high concentrations of Cremophor EL did reduce the rate of uptake of paclitaxel into cells but had little effect on total paclitaxel accumulation. These results confirm that paclitaxel has striking effects on the cell cycle and show that high concentrations of Cremophor EL are capable of inducing a cell cycle block distinct from the mitotic block seen with paclitaxel. These results also demonstrate that cells exposed to paclitaxel for longer than 24 hours attempt to complete mitosis but the process of cytokinesis is inhibited. Together with cytotoxicity data, these results indicate that entry into and exit out of mitosis are prerequisites for paclitaxel cytotoxicity.

Schweinfurthin A Selectively Inhibits Proliferation and Rho Signaling in Glioma and Neurofibromatosis Type 1 Tumor Cells in a NF1-GRD–Dependent Manner

Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1−/+;Trp53−/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1−/−;Trp53−/− astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor–stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling. Mol Cancer Ther; 9(5); 1234–43. ©2010 AACR.

Association Energetics of Cross-Reactive and Specific Antibodies†

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

Investigation Of A 6-fluorotryptophan Substituted scFv

For many years our laboratory has pursued an understanding of the protein characteristics which confer specificity and affinity to the antibody for its antigen using a family of monoclonal antibodies to hen egg white lysozyme (HyHEL26, 10, 8 and 63, primarily.) We find that the binding is best characterized by a two-step model representing an association complex becoming a docked complex, evidencing a conformational change.In a recently produced scFv variant of HyHEL10 in which all the tryptophans were substituted with the 6-fluoro form we studied kinetic behavior by Biacore SPR, using our usual protocol to obtain kinetic characterization. We observed that the affinity to lysozyme was concentration dependant, though it did not reflect oligomerization; it changes gradually, allowing investigation, decreasing by an order of magnitude over a period of 3 hours and that most of the change is due to the decrease in the docking step. This repeatable behavior is reversed upon sample reconcentration and delayed by cold. To explore the possible role of folding or water movement we investigated the impact of TMAO, glycerol and some detergents. We also did further exploration by SPR, fluorescence spectroscopy, and other biophysical characterizations in order to better understand the molecular events responsible for this dramatic affinity change.