Taxol inhibited HeLa cell proliferation by inducing a sustained mitotic block at the metaphase/anaphase boundary. Half-maximal inhibition of cell proliferation occurred at 8 nM taxol, and mitosis was half-maximally blocked at 8 nM taxol. Inhibition of mitosis was associated with formation of an incomplete metaphase plate of chromosomes and an altered arrangement of spindle microtubules that strongly resembled the abnormal organization that occurs with low concentrations of vinblastine and other antimitotic compounds. No increase in microtubule polymer mass occurred below 10 nM taxol. The mass of microtubules increased half-maximally at 80 nM taxol and attained maximal levels (5 times normal) at 330 nM taxol. At submicromolar concentrations, taxol suppressed growing and shortening at the ends of microtubules reassembled in vitro from bovine brain tubulin in a manner that resembled suppression by vinblastine. Taxol was concentrated in HeLa cells several hundredfold to levels that were similar to those which suppressed dynamic instability in vitro. The results indicate that taxol shares a common antiproliferative mechanism with vinblastine. At its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

The antiproliferative action of vinblastine at low concentrations appears to result from modulation of the polymerization dynamics of spindle microtubules rather than from depolarization of the microtubules [Jordan, M. A., Thrower, D., & Wilson, L. (1991) Cancer Res. 51, 2212-2222; (1992) J. Cell. Sci. 102, 401-416]. In the present study, we used differential interference contrast video microscopy to analyze the effects of vinblastine on the growing and shortening dynamics (dynamic instability) of individual bovine brain microtubules in vitro. With microtubules which were either depleted of microtubule-associated proteins (MAPs) or rich in MAPs, low concentrations of vinblastine (0.2 microM-1 microM) suppressed the growing and shortening rates and increased the percentage of time that the microtubules spent a state of attenuated activity, neither growing nor shortening detectably. Vinblastine also suppressed the duration of microtubule growing and shortening, and increased the duration of the attenuated state, during which the microtubules neither grew nor shortened detectably. Consistent with previous data obtained using radiolabeled nucleotide exchange in microtubule suspensions [Jordan, M. A., & Wilson, L. (1990) Biochemistry 29, 2730-2739], vinblastine suppressed growing and shortening dynamics at the kinetically more rapid plus ends. The results suggest that vinblastine kinetically stabilizes microtubule ends by modulating the gain and loss of the stabilizing GTP or GDP-Pi "cap", which is believed to be responsible for the transitions between the growing and shortening phases. The data support the hypothesis that (1) low concentrations of vinblastine inhibit mitosis by kinetically stabilizing the polymerization dynamics of spindle microtubules and that (2) the dynamics of spindle microtubules are critical for the proper progression of mitosis.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....

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