No abstract

Hydropathy analysis of the amino acid sequence of the Pho84 phosphate permease of Saccharomyces cerevisiae suggests that the protein consists of 12 transmembrane domains connected by hydrophilic loops. The Pho84 protein has been modified by a gene fusion approach, yielding two different Nterminal His-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes. The continuous epitopes in the N-and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 proteins. Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into liposomes.z 1999 Federation of European Biochemical Societies.

The insulin-like growth factor-1 receptor (IGF-1R) mitogenic signaling mediates malignant cell survival by many complex and redundant pathways. This study compared the effects of IGF-1R inhibition on viability and apoptosis of two NSCLC cell lines, using three different methods for the impairment of IGF-1R function: (IR3, an anti-IGF-1R antibody; tyrphostin AG1024, a tyrosine kinase inhibitor (TKI) and IGF-1R-small interfering RNA (siRNA). IGF-1R inhibition led to a decrease of cell survival and induced apoptosis in a manner depending on the approach used for the receptor inhibition. To find an explanation, we analyzed the effects of these treatments on three major antiapoptotic pathways evoked by IGF-1R signaling: IRS-1, Shc and 14.3.3-dependent mitochondrial translocation of Raf-1 kinase (mitRaf). (IR3 downregulated IRS-1 phosphorylation in A549 cells and Shc phosphorylation in U1810 cells. While in A549 cells AG1024 treatment decreased both IRS-1 and Shc phosphorylation, in U1810 cells the IRS-1 phosphorylation was only slightly affected and the Shc phosphorylation drastically downregulated. Neither (IR3 nor AG1024 had any effect on Raf-1 kinase translocation. Irrespective of the cell line, IGF-1R-siRNA treatment induced downregulation of both IRS-1 and Shc phosphorylation coupled with the abrogation of mitRaf. In addition, the IGF-1R-siRNA proved to be the most potent inducer of apoptosis suggesting that more than one antiapoptotic pathway in IGF-1R signaling should be inhibited to effectively induce apoptosis in lung cancer cells.

The restriction point (R) separates the G1 phase of continuously cycling cells into two functionally different parts. The first part, G1-pm, represents the growth factor dependent post-mitotic interval from mitosis to R, which is of constant length (3-4 h). The second part, G1-ps, represents the growth factor independent, pre-S phase interval of G1 that lasts from R to S and that varies in time from 1 to 10 h. G1-pm cells rapidly exit (within 1 h) from the cell cycle and enter G0 as a response to serum withdrawal. The finding that R occurs at a set time after mitosis indicates that R may be related to the metabolic and/or structural changes that the cell underwent during the previous mitosis. We have recently shown that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is not the molecular mechanism behind R, as has been suggested previously. Here, we present an alternative explanation for R. In the present study, we applied a single cell approach using time-lapse analysis, which revealed that upon serum starvation the G1-pm cells rapidly underwent a transient change in cell shape from flat to spherical before exiting to G0. Platelet derived growth factor (PDGF) counteracted this change in shape and also prevented exit to G0 to the same extent. Furthermore epidermal growth factor (EGF) and insulin like growth factor (IGF-1), which only partially counteracted this change, only partially counteracts exit to G0. These data clearly indicate a direct link between change in cell shape and exit to G0 in G1-cells that have not passed R.