A phase II study of docetaxel in patients with metastatic squamous cell carcinoma of the head and neck

This study was designed to evaluate the activity, safety and tolerance of docetaxel (D) in a selected population with metastatic squamous cell carcinoma of the head and neck (SCCHN). Twenty-four patients with no prior palliative therapy were enrolled and received D 100 mg m−2 by 1 h of infusion, every 3 weeks. All but two patients had been evaluated for efficacy on lung metastatic sites. No prophylactic administration of anti-emetics or growth factors was given. A pharmacokinetic study was performed in 22 patients. Twenty-one patients were assessable for response and 24 for toxicity. One hundred and four cycles were administered with a median of 4.5 (range 1–9) per patient. The median cumulative dose was 449 mg m−2. Partial responses were achieved in five patients with a median duration of 18.7 weeks (range 13.1–50.3). The overall response rate was 20.8% with a median duration of 11.0 weeks (range 2.4–52.6). The most frequent side-effect was neutropenia (79.2% grade IV) but with a short duration (median 4 days) and no febrile neutropenia. The incidence of moderate/severe fluid retention was 29.2% with one treatment discontinuation. Other toxicities (all grades) were common (skin 75%, asthenia 50%, infection 29.2%, nausea 16.7%, diarrhoea 12.5%, stomatitis 16.7%, vomiting 8.3% and HSR 8.3%). A mean clearance of 19.6 l h−1 m−2 and an area under the curve of 6.00 μg ml−1 h−1 was found in the pharmacokinetic analysis. Docetaxel is active in this selected population with metastatic SCCHN, with a good tolerance. © 1999 Cancer Research Campaig

Biophysical characterization of interactions involving importin-alpha during nuclear import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K-D = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K-D = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K-D = 1.7 x 10(-8) m; nucleoplasmin NLS, K-D = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import

Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Schedule-dependent cytotoxicity of SN-38 in p53 wild-type and mutant colon adenocarcinoma cell lines

In this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml–1 with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50–100 ng ml–1). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration (P < 0.001, P = 0.01 respectively), whereas in S and G2/M p53 expression increased only with time (P < 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure × duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53. © 1999 Cancer Research Campaig

UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM