Monika Vetterlein

Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells

Basic fibroblast growth factor (FGF‐2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF‐2 and FGF receptors (FGFR‐1 to ‐4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF‐2 and FGFR in vitro and in vivo. FGF‐2 expression did correlate with a short doubling time as well as with potent anchorage‐independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF‐2 into the extracellular space. Expression levels of FGFR‐1 and ‐2 in NSCLC cell lines correlated with FGF‐2 production. FGFR were located at the plasma membranes in some low FGF‐2‐producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF‐2 (rFGF‐2). In NSCLC cell lines with an enhanced FGF‐2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF‐2 and growth inhibition by FGF‐2‐neutralising antibodies. In contrast, several agents antagonised FGF‐2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF‐2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism. Int. J. Cancer 83:415–423, 1999.

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.

Possible role of the multidrug resistance‐associated protein (MRP) in chemoresistance of human melanoma cells

Human malignant melanoma is characterised by unresponsiveness to conventional chemotherapy. Melanoma‐derived cell lines are often markedly chemoresistant, suggesting that cellular mechanisms mediate the multidrug resistance (MDR) phenotype. The multidrug resistance‐associated protein (MRP) is a drug transporter protein associated with resistance to a broad spectrum of lipophilic drugs. To investigate whether MRP is involved in intrinsic drug resistance of human melanoma, we analysed expression and functional activity of MRP as well as its impact on chemoresistance in 40 melanoma cell lines (35 established by us from primary and metastatic lesions and 5 obtained from international sources), as well as in one dysplastic naevus‐derived cell line and in normal melanocytes. By reverse transcriptase‐polymerase chain reaction various levels of MRP mRNA were detected in all melanoma cell lines, and by immunoblot the corresponding protein in a high percentage of them. Functional activity of MRP was assayed by analysing cellular accumulation of 3H‐daunomycin (3H‐DM) and calcein in response to MRP‐modulators by β‐spectrometric and fluorescence‐activated cell sorter analysis, respectively. Probenecid (PRO), N‐eth‐ylmaleimide (NEM) and benzbromarone (BB) moderately (≤ 1.43‐fold) but significantly enhanced intracellular accumulation of MRP substrate probes corresponding to MRP expression. Moreover, the sensitivity of melanoma cell lines to daunomycin (DM) and doxorubicin (DOX), but not to vinblastine (VBL), etoposide (VP‐16) and cisplatin (CDDP), analysed by an MTT‐based survival assay, were inversely correlated with MRP‐gene expression. Our results imply that MRP may be a component of the intrinsic chemoresistance phenotype characteristic of human malignant melanoma. Int. J. Cancer, 71:108–115, 1997.

Endocytic routes to the Golgi apparatus.

Abstract The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

Detection of adrenoleukodystrophy by increased C26:0 fatty acid levels in leukocytes.

Very long chain fatty acids of peripheral blood leukocytes were analyzed by gas chromatography in nine members of a family including two hemizygotes and one obligate heterozygote for adrenoleukodystrophy (ALD), as well as in twelve controls. Comparative investigations were done in cultured fibroblasts. Elevated C26:0 levels were observed in leukocytes and fibroblasts of ALD hemizygotes. The obligate heterozygote displayed a clear-cut increase of C26:0 concentration in leukocytes but not in fibroblasts. Determination of C26:0 in leukocytes may serve as diagnostic tool in the detection of ALD gene carriers.

Expression of myc and ras oncogenes in two newly established neuroblastoma cell lines

SummaryTwo new neuroblastoma cell lines, KG-MH and KM-YH have been established from fresh tumour samples. In vitro growth characteristics are presented together with a karyological analysis. Northern and Southern blot experiments have been performed using molecularly cloned probes for c-myc, N-myc, c-Ha-ras, c-Ki-ras, and N-ras oncogenes. Both cell lines showed expression for N-myc, while c-myc expression was not detected. Cell line KM-YH, with a rather long population doubling time of 78 h, showed additional expression for the three ras genes.

Unterschiede der in vitro-Migration von kultivierten Melanomzellen

Eines der spezifischen Charakteristika maligne entarteter Tumoren ist das lokal destruktive Wachstum sowie die Tendenz zur Bildung von fernmetastatischen Absiedelungen. Diese Ausbreitungstendenz der Zellen aus Primartumoren kann wahrscheinlich durch sehr unterschiedliche Mechanismen, die sowohl vom Wirtsorganismus als auch durch die Tumorzellen selbst induziert sind, unterstutzt werden.