Synthesis and secretion of plasminogen activators and plasminogen activator inhibitors in cell lines of different groups of human lung tumors.

Several human lung tumor cell lines derived from large cell, squamous cell, and small cell carcinomas, as well as from mesotheliomas of the lung have been investigated for their gene expression and secretion of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitors 1 and 2. All bronchogenic non-small cell carcinoma-derived cell lines studied could produce either plasminogen activators, their inhibitors, or both components, whereas in small cell lung carcinoma cell lines and cell lines derived from mesothelioma of the lung, no substantial amounts of any of these substances were synthesized. In detail, a large cell carcinoma-derived cell line, LCLC 97TM1, constitutively secreted large amounts of plasminogen activator. Northern blot analysis revealed RNA specific for u-PA and t-PA. Another large cell carcinoma-derived cell line, LCLC 103H, secreted smaller amounts of plasminogen activator and, additionally, plasminogen activator inhibitor. Specific mRNAs for u-PA and plasminogen activator inhibitors 1 and 2 were found in this cell line. In contrast, squamous cell carcinoma-derived cell lines secreted plasminogen activator only after treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate; enhanced levels of u-PA, t-PA, and plasminogen activator inhibitor 1 mRNAs could then be demonstrated. The different expression of the plasminogen activator enzyme system distinguishes cell lines derived of non-small cell lung carcinoma from those of small cell lung carcinoma and may also reflect significant differences in the biological behavior of these tumor types.

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi.

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.

The complete nucleotide sequence of the duck alpha A-globin gene.

We report the entire nucleotide sequence of the duck alpha A-globin gene including 330 nucleotides of the 5'-flanking region. The amino acid coding sequence of the 815-bp-long gene is interrupted at preserved locations by two intervening sequences. In contrast to all other vertebrate globin genes investigated so far, the first intervening sequence (150 bp) of the duck alpha A-globin gene exceeds the size of the second intervening sequence (104 bp) by 46 bp. Moreover, the alpha A-globin gene has an extraordinary GC-rich 5'-flanking region. Conserved regions are identified which might encode signals for transcriptional initiation, RNA splicing and poly(A) addition.

The isolation and partial characterization of linked alpha A- and alpha D-globin genes from a duck DNA recombinant library.

Genomic DNA from an adult duck (Cairina moschata) was used to construct a library of cloned DNA fragments in the vector lambda Charon 4A. Screening of the DNA library resulted in the isolation of a recombinant, D alpha G-1, which carries both the adult duck alpha A- and alpha D-globin genes. The two globin genes are separated by approx. 2.2 kb of DNA, they are encoded by the same DNA strand and their orientation with respect to the direction of transcription is 5'- alpha D- alpha A-3'. Partial sequence analyses indicate that the two alpha-globin genes contain intervening sequences at positions homologous to those in chicken and mammalian alpha-globin genes.

Genomic organization and primary structure of five homologous pairs of intron-less genes encoding secretory globins from the insect Chironomus thummi thummi.

From a Chironomus thummi thummi genomic library we have isolated two distinct recombinant phages, CttG-1 and CttG-3, each carrying a cluster of five homologous globin genes. In addition to the previously reported nucleotide sequence of globin gene D (Antoine and Niessing, 1984) we present the chromosomal arrangement, primary structure and predicted amino acid sequence of nine globin genes. The divergently transcribed globin genes all lack introns, they encode secretory preglobins each containing a highly conserved signal peptide. The amino acid sequences deduced from the globin genes correspond to globin III and variants thereof, to globin IV, and to a novel globin, whose direct amino acid sequence has not yet been reported.

Different pattern of expression of cellular oncogenes in human non-small-cell lung cancer cell lines.

Altered and deregulated cellular oncogenes were found in many human solid tumors. Except for a few types of tumors that consistently exhibited specific altered proto-oncogenes, the majority of tumors are associated with a number of transcriptionally activated cellular oncogenes. In the heterologous group of non-small-cell lung cancer (NSCLC), nothing about a specific pattern of proto-oncogene expression is known. Therefore, we investigated the expression of a panel of cellular oncogenes in NSCLC cell lines. DNA and RNA from 11 established NSCLC cell lines (4 adenocarcinoma cell lines, 3 squamous cell carcinoma cell lines, 3 large-cell carcinoma cell lines and 1 mesothelioma cell line) were isolated and analysed using the Southern, dot blot and Northern hybridization technique. c-myc RNA expression was found in all NSCLC cell line, L-myc expression only in 1 adenocarcinoma cell line, N-myc and c-myb expression in none of the 11 cell lines examined. No c-myc amplification could be detected in the DNAs. v-sis-related mRNA was observed in 5/11 cell lines without association to a specific NSCLC subtype. v-src-related mRNA, found in all tested cells, exhibited increased levels in 1 adenocarcinoma cell line (A-549) compared to the other cell lines. Binding sites for epidermal growth factor (EGF) had been described previously in NSCL, therefore we found erbB homologue transcripts coding for the EGF receptor in all NSCLC cell lines. Also, c-raf1-, N-ras-, Ki-ras-, and H-ras-related RNA expression was observed in all lines. We conclude that L-myc, N-myc, and c-myb expression does occur less frequently in NSCLC than in SCLC. Also amplification does not appear to be an important mechanism by which the c-myc proto-oncogene is activated in NSCLC. A specific pattern of oncogene expression could not be detected in NSCLC cells; each cell line examined showed its own pattern. However, transcriptional activation of a proto-oncogene like erbB, ras, raf, src, and c-myc, which are all involved in the progression pathway of EGF, may be a common feature of NSCLC.

Characterization of insulin-like growth factor I receptors and growth effects in human lung cancer cell lines.

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell lines were studied for insulin-like growth factor I (IGF-I) receptor expression and with regard to the influence of IGF-I on cell proliferation. IGF-I receptors on the cells were characterized by competitive binding assays, chemical crosslinking and northern blot hybridization of IGF-I receptor mRNA. All SCLC and NSCLC cell lines showed specific IGF-I binding sites with an affinity (KD) of 0.69-5.21 nM. The amount of binding sites ranged from 59 fmol/mg to 1230 fmol/mg protein. The IGF-I binding was inhibited by the IGF-I receptor antibody (alpha-IR-3). Northern blot hybridization indicated that IGF-I receptor mRNA was being produced by all SCLC and NSCLC cell lines. We used the soft-agarose clonogenic assay to evaluate the influence of IGF-I on the in vitro proliferation of the cells. Our results have shown that IGF-I stimulates the growth of all tested cell lines ranging from a factor of 1.6 to 4.2 in SCLC and from 1.1 to 2.7 in NSCLC. The data indicate that the IGF-I receptor thus appears to be the common pathway for the mitogenic activity of IGF-I and IGF-II with regard to human lung cancer cells.

Growth regulation by insulin-like growth factors in lung cancer.

Lung cancer is a major health problem, with over 38,000 new cases expected every year in West Germany. A more complete understanding of the biology of lung cancer will hopefully lead to therapeutic modalities. The possible autocrine growth regulation in small-cell lung cancer and non-small-cell lung cancer has been demonstrated for bombesin/GRP, vasopressin, neurotensin, EGF/TGF alpha, transferrin-related peptides and insulin-like growth factors. This contribution concentrates on recent data concerning binding sites, growth promoting effects and secretion of IGFs in lung cancer cell lines. The production of IGF-binding proteins which were also produced by lung cancer cell lines modifies the autocrine/paracrine model for IGFs since then proteins can either enhance or inhibit the effect of IGFs on tumor growth.

Determination of copper, chromium, manganese and zinc by graphite furnace atomic absorption spectrometry after separation on polyacrylamide modified with nitrilo triacetic acid.

A new chelating collector, polyacrylamide modified with nitrilo triacetic acid (NTA) was developed for the separation and preconcentration of copper, chromium, manganese, and zinc prior to their determination by Graphite Furnace Atomic Absorption Spectrometry (GFAAS). The retention and recovery of the analyte elements were investigated by applying batch and column techniques. Cu(II), Cr(III), Mn(II) and Zn(II) were quantitatively retained by the collector at pH 5.5 or above. The chelating kinetics are so fast that in the batch procedure a quantitative separation of the analyte elements can be achieved in a few seconds. Since a very short contact time is enough to retain the analyte elements in column technique, a separation step can be completed quickly by applying fast flow rates in small columns. The elements collected were completely recovered with 2 mol/l of HCl. In the presence of sodium chloride up to 0.5% the analyte elements were quantitatively separated and recovered. Low blank values of the collector is another important advantage.

The primary structure of the duck alpha D-globin gene: an unusual 5' splice junction sequence.

The complete nucleotide sequence of the duck minor alpha D-globin gene including the flanking regions has been determined. A unique structural feature of the alpha D-globin gene is a GC instead of the invariant GT dinucleotide at the 5' end of the second intervening sequence. The 1013 base pair long gene has otherwise all the characteristics normally attributed to a functional globin gene. Indirect evidence suggests that the alpha D-globin gene is expressed in vivo.

Characterization of insulin-like growth factor II receptors in human small cell lung cancer cell lines.

Small cell lung cancer (SCLC) cell lines were investigated for the expression of insulin-like growth factor (IGF) II receptor by means of radioreceptor assays, cross-linking techniques, and Northern blot analysis. 125I-IGF II binds to both the IGF I and the IGF II receptor on intact SCLC cells. Detailed receptor assays performed on microsomal and plasma membranes gave evidence that 125I-IGF II binds with high affinity (70-80 pM) to the IGF I receptor and with low affinity (2-4 nM) to the IGF II receptor, and not conversely. The presence of mannose-6-phosphate enhanced the binding of 125I-IGF II to the IGF II receptor of SCLC. Mannose-6-phosphate also increased the efficiency of N-hydroxysuccinimide ester in cross-linking 125I-IGF II to the IGF II receptor and facilitated the cross-linking of 125I-IGF II to a second protein of 240-250 kDa. Soluble IGF II receptor was also detected in conditioned media of SCLC cell lines.