Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma.

INTRODUCTION: Cytokines of the gp130-family, particularly interleukin(IL)-6, play a crucial role in the propagation of malignant plasma cells. MATERIALS AND METHODS: The role of IL-6 and other gp130-cytokines was studied in the human plasma cell line INA-6 in vitro and in INA-6 xenografts. The proliferative response to gp130-cytokines was evaluated and activated components of gp130-signaling pathways were identified by Western blotting and DNA binding studies. Specifically, expression of IL-6 and receptors for IL-6 and leukemia inhibitory factor were analysed by RT-PCR and ELISA. RESULTS: The plasma cell line INA-6 was cultured for several years remaining strictly dependent on exogenous IL-6. Other gp130-cytokines had no significant effect on INA-6 cell proliferation in vitro. Due to an activating mutation in the N-ras gene, mitogen-activated protein kinases (MAPK) were constitutively phosphorylated. In contrast, signal transducer and activator of transcription(STAT)-3 activation was dependent on stimulation with IL-6. Blocking of either one of these pathways resulted in a significant decrease of INA-6 cell proliferation. Remarkably, INA-6 xenografts did not require exogeneous IL-6 for proliferation in vivo. Instead, an autocrine IL-6 loop and, in certain tumor sublines, responsiveness to additional gp130-cytokines was induced during in vivo growth. CONCLUSION: Activation of the gp130 signal transducer is mandatory for INA-6 cell growth in vitro and in vivo. Both the MAPK and the Jak/STAT pathway are operative in malignant plasma cells and either one is essential for plasma cell growth. The INA-6 cell line provides a preclinical model to study growth regulation of human plasmacytoma cells and to evaluate novel therapeutic strategies.

Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea (Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.