In situ analysis of the action of Navelbine on various types of microtubules using immunofluorescence.

Preliminary clinical studies demonstrated that 5' nor-anhydro-vinblastine, Navelbine (NVB) has a broader antitumor activity and fewer neurotoxic effects than vinblastine or vincristine. The tectal plate anlage of mouse embryos at the earliest stages of neuronal differentiation were used to analyze and compare the effect of NVB, vincristine and vinblastine on axonal and mitotic microtubules after culture of post-implantation embryos in a medium containing the agent. All drugs are active on mitotic microtubules at the same concentration (0.1 mumol/L), inducing a depolymerization of microtubules and a blockade of cells at metaphase. At higher concentrations. NVB is the only one of the three drugs that induces a blockade of the cells at prophase. A depolymerization of axonal microtubules occurs at higher concentrations with NVB than with the two other vinca alkaloids. These results demonstrate that NVB is as active on mitotic microtubules and less active on axonal microtubules than vincristine and vinblastine. These findings can be related to the potent antitumor effect of the drug with minor neurotoxicity.

Biochemical effects of Navelbine on tubulin and associated proteins.

Navelbine (NVB) or 5' nor-anhydro-vinblastine was shown to present a broader antitumor activity and to induce fewer side effects than vinblastine (VBL) or vincristine (VCR). The possible mechanisms of these differences were analyzed with in vitro methods. At substoichiometric concentrations, the three drugs inhibit microtubule assembly. NVB, in comparison with VCR and VBL, is shown to have a lower inhibitory effect. At stoichiometric concentrations, the three drugs are able to induce tubulin aggregation into spirals and paracrystals. This process involves a microtubule-associated protein (MAPs) family referred to as Tau and is inhibited by another MAPs family referred to as MAP2. However, dramatic quantitative and qualitative differences are observed between NVB and VLB or VCR in TAU-induced aggregation of tubulin. The rate and extent of NVB-induced tubulin aggregation is much lower. With NVB, only certain TAU isoforms are able to induce paracrystals, while all TAU isoforms may contribute to VCR-induced or VBL-induced paracrystals. The TAU isoforms that are not able to induce crystallization with NVB, at least in a certain range of concentrations, are probably involved in mitotic microtubules--the hypothetical antitumoral target of vinca alkaloids (VAS). The present work shows for the first time that an anticancer drug is able to discriminate between the various types of microtubules. A next step will be to investigate whether this property is limited to a modulating effect of the various TAU isoforms on the affinity of VAS for tubulin. These biochemical investigations will be extended to tubulins extracted from tumor cell lines in order to further discriminate NVB from the other VAS.

Physical and genetic characterization of deletions in Streptococcus pneumoniae.

Genetic properties of markers may discriminate between deletions and point mutations. We have designed a physical method for a direct characterization of deletions which also gives an estimate of their size.

Relation between the transforming activity of a marker and its proximity to the end of the DNA particle.

Transforming pneumococcal DNA is inactivated by treatment with restriction enzymes. For mutations belonging to the same locus (amiA locus), the extent of inactivation depends strongly upon the mutations and the enzymes. Two EcoRI and one BamHI restriction sites have been located within the amiA locus. After treatment of donor DNA with either one of these enzymes, the lowest transforming activity is observed for mutations that map near restriction sites. This effect of proximity to the nearest end of the DNA fragment extends over a distance of 1,400 nucleotides. The curve of transforming activity versus DNA size obtained with endonuclease-generated DNA fragments is very similar to that obtained previously with mechanically sheared DNA. Both curves show a striking slope change for donor DNA size around 2,700 base pairs, i.e. twice the length found for the extent of the 'end effect'. We suggest that for donor DNA fragments larger than 2,700 base pairs the transforming activity depends mainly upon the size of donor whereas for donor DNA fragments shorter than 2,700 base pairs both a size-dependent phenomenon and the 'end effect' contribute to reduce drastically the transforming activity.

Relationships between the structures of taxol and baccatine III derivatives and their in vitro action on the disassembly of mammalian brain and Physarum amoebal microtubules.

The in vitro disassembly of microtubules from mammalian brain and Physarum is inhibited by various derivatives of taxol and baccatine III. Structure-activity relationships of the taxol derivatives were identical for both mammalian brain and Physarum microtubules. This observation suggests that the site of action of taxol has been preserved during the evolution of these two different eukaryotic lines. The substituent at C-13 of taxol was required to prevent disassembly of brain microtubules with or without microtubule-associated proteins. In contrast, both taxol and baccatine III prevented the disassembly of Physarum microtubules to the same extent, showing that the substituent at C-13 was not required in the interaction with Physarum tubulin. The different effects of baccatine III and taxol derivatives indicate that measuring the disassembly of microtubules from different organisms could be a useful parameter in the search for derivatives exhibiting antiparasitic activity.

Immunofluorescence study of the action of navelbine, vincristine and vinblastine on mitotic and axonal microtubules.

Among the various non-naturally-occurring Vinca alkaloid compounds, nor-anhydro-vinblastine (Navelbine, NVB) exhibits in preliminary clinical studies broader anti-tumor activity and lower neurotoxicity than vinblastine (VBL) and vincristine (VCR). The action of these 3 Vinca alkaloids on axonal and mitotic microtubules has been studied experimentally in a specific model, the tectal plate anlage of mouse embryos at the earliest stages of neuronal differentiation. Post-implantation embryos were cultured in toto in a medium containing increasing concentrations of drugs. Microtubules were stained using immunofluorescence with a tubulin-specific polyclonal antibody in semi-thin sections after embedding in high-molecular-weight polyethylene glycol. All drugs induced depolymerization of mitotic interpolar microtubules and cell metaphase block at the same concentration. Increasing the concentrations led to progressive depolymerization of kinetochore microtubules. However, NVB was the only drug to induce complete microtubule depolymerization. The activity of the 3 compounds on axonal microtubules was identical: depolymerization of a labile pool of microtubules. This was observed at higher concentrations with NVB than with the 2 other Vinca alkaloids. Our results show that, in this model, NVB is as active on mitotic microtubules as VCR and VBL, and less active on axonal microtubules. None of the 3 drugs modified microtubule length but all appeared to induce disruption of the labile microtubule pool without altering the stable pool.