A crucial function of PDGF in TGF-beta-mediated cancer progression of hepatocytes.

Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

Xenogenization by tetanus toxoid loading into lymphoblastoid cell lines and primary human tumor cells mediated by polycations and liposomes.

We explored the potential of the xenogenization concept as an adjuvant procedure in anti-tumor immunity. To mediate effective loading we used polyarginine (pArg) molecules of various degrees of polymerization, cationic liposomes, or chimeric molecules of transferrin (Tf) and the polycation polyethyleneimine (PEI). Tetanus toxoid (TT) was loaded onto primary human leukemia cells, culture adapted primary human neuroblastoma cells, and human lymphoblastoid cell lines (LCLs) with high efficiency by all procedures. Trypsin treatment of loaded cells provided evidence that only liposomes and Tf-PEI mediated internalization of TT. Lymphocytes primed with xenogenized LCLs and challenged with unmodified LCLs showed increased IFNgamma secretion compared with lymphocytes primed with non-xenogenized LCLs.

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.

Subunit composition and quantitative importance of hetero-oligomeric receptors: GABAA receptors containing alpha6 subunits.

In cerebellum, GABAA receptors containing alpha6 subunits are expressed exclusively in granule cells. The number of alpha6 receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on alpha6 subunit-specific antibodies indicated that 45% of GABAA receptors in cerebellar extracts contained alpha6 subunits. Western blot analysis demonstrated that alpha1, beta1, beta2, beta3, gamma2, and delta subunits co-purified with alpha6 subunits, suggesting the existence of multiple alpha6 receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of receptors containing the co-purifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of alpha6 receptors remaining in the extract, the proportion of alpha6 receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that alpha6 receptors in cerebellum are composed predominantly of alpha6betaxgamma2 (32%), alpha1alpha6betaxgamma2 (37%), alpha6betaxdelta (14%), or alpha1alpha6betaxdelta (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of alpha6 receptors contained homogeneous beta1, beta2, or beta3 subunits, respectively, whereas two different beta subunits were present in 18% of all alpha6 receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABAA receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.