Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells.

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.

Multiple tyrosine protein kinases in rat hippocampal neurons: isolation of Ptk-3, a receptor expressed in proliferative zones of the developing brain.

Tyrosine protein kinases are likely to play an important role in the maintenance and/or development of the nervous system. In this study we have used the PCR cloning technique to isolate sequences derived from tyrosine kinase genes expressed in cultured hippocampal neurons obtained from 17.5-day-old rat embryos. Nucleotide sequence analysis of 209 independent clones revealed sequences derived from 25 tyrosine kinases, of which two corresponded to previously unreported genes. One of the PCR clones, ptk-2, belongs to the Jak family of cytoplasmic tyrosine kinases. The second clone, ptk-3, was derived from a gene encoding an additional class of tyrosine kinase receptors whose extracellular domains contain regions of homology with coagulation factors V and VIII and complement component C1. Transcripts encoding the Ptk-3 receptor are present in a variety of embryonic and adult tissues with highest levels observed in brain. During development, ptk-3 transcripts are most abundant in the proliferative neuroepithelial cells of the ventricular zone, raising the possibility that this receptor may play an important role in the generation of the mammalian nervous system.

vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells.

A novel human oncogene, designated vav, was generated by a genetic rearrangement during gene transfer assays. The vav oncogene directs the synthesis of a 3.0 kb mRNA from which we isolated a 2.8 kb-long complementary DNA copy. Nucleotide sequence analysis of this vav oncogene cDNA clone revealed that its 5' 167 bp were derived from pSV2neo DNA cotransfected as a selectable marker during gene transfer. The remaining 2597 bp were unrelated to genes included in current data banks, indicating that the vav oncogene is likely to be derived from a novel human locus. The vav oncogene cDNA clone encompasses a 2391 bp long open reading frame (ORF) capable of directing the synthesis of a 797 amino acid long polypeptide. The predicted vav oncogene protein sequence exhibits several motifs reminiscent of transcriptional factors. They include a highly acidic amino-terminal region separated from two putative nuclear localization signals by a proline-rich sequence, presumably a hinge region. In addition, we identified two zinc-finger-like domains, one of which conforms to the canonical pattern Cys-X2-Cys-X13-Cys-X2-Cys previously found to confer trans-activating activity to the adenovirus E1A protein. Transcription of its normal allele, the vav proto-oncogene, has been exclusively observed in cells of hematopoietic origin, including those of erythroid, lymphoid and myeloid lineages. These findings raise the possibility that this novel locus might play an important role in hematopoiesis.

A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences.

A biologically active complementary DNA clone of a transforming gene present in a human colon carcinoma contains gene sequences of both tropomyosin and a previously unknown protein tyrosine kinase. The predicted protein (641 amino acids) encoded by this oncogene seems to have been formed by a somatic rearrangement that replaced the extracellular domain of a putative transmembrane receptor by the first 221 amino acids of a non-muscle tropomyosin molecule.

Inhibitors of polypeptide elongation on yeast polysomes.

Yeast polysomes are very active for amino acid incorporation when supplemented with elongation factors and the different components required for elongation of the polypeptide chain. This polysomal system is suitable for the study of the individual streps of the elongation cycle and to test the effect of different inhibitors. Anisomycin, trichodermin, trichodermol, trichothecin, fusarenon X, sparsomycin and blasticidin S inhibit peptide bond formation on these polysomes, whereas diphtheria toxin, pederine, cycloheximide and cryptopleurine block translocation.