Gabriele Jaques

Establishment, growth properties, and morphological characteristics of permanent human small cell lung cancer cell lines

SummaryCell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in estabilishing and permanently growing SCLC. Serum-free SIT medium and SIT 2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R10 medium was suprior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.

Peptides and growth factors in small cell lung cancer: production, binding sites, and growth effects

SummaryWe investigated the production, binding to cell membranes, and influence on cell proliferation of peptides and growth factors in 4 classic, 5 transitional and 5 variant SCLC cell lines. Glucagon, neurotensin, and TGF-α were present in all cell lines. Bombesin was predominantly found in classic cell lines and insulin in variant cell lines. Neurokinin A, calcitonin, CGRP, GHRF, somatostatin, and CNTF were detectable in some cell lines without prevalence for a particular cell type. We could not detect AVP, growth hormone, neuropeptide Y, substance P, VIP, and NGF. Insulin binding sites were present on 11/14 cell lines, and some cell lines specifically bound bombesin, calcitonin, and EGF. Growth effects were detectable for insulin, GRP-related peptides, tachykinins, and VIP. Using serum-free conditions, insulin and VIP had a growth stimulating effect in liquid culture at nanomolar concentrations. Bombesin and neuromedin B stimulated the clonal growth at a concentration of 3–30 nM. The tachykinins neurokinin A, neurokinin B, physalaemin, and eledoisin inhibited the clonal and mass culture growth with a peak effect in the range of 0.1 to 10 pM. Peptide-induced stimulating and inhibiting effects were within a magnitude of 2-fold. All other peptides and growth factors tested, including ACTH, AVP, calcitonin, glucagon, neurotensin, somatostatin, EGF, CNTF, and NGF did not affect the growth of SCLC. We conclude that the growth of SCLC is partly controlled by such peptides in an autocrine/paracrine fashion.

Production of immunoreactive insulin-like growth factor I and response to exogenous IGF-I in small cell lung cancer cell lines

Small cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth factor I-related protein (IGF-I) in cell pellets and culture media. IGF-I immunoreactivity was detected in 11/14 pellets, ranging from 12 to 76 mIU/mg soluble protein. The IGF-I levels in the cell pellets showed a correlation to the corresponding culture media. IGF-I binding sites were found in all tested cell lines. The maximum binding (Bmax) ranged from 131 to 1230 fmol/mg protein and the dissociation constant (KD) from 0.89 to 5.21 nM. The incorporation of [3H]thymidine in the presence of recombinant human IGF-I resulted in a clearly increased DNA synthesis in two of seven cell lines. Thus, IGF-I may be an important growth factor in SCLC.

Prognostic value of pretreatment carcinoembryonic antigen, neuron-specific enolase, and creatine kinase-BB levels in sera of patients with small cell lung cancer

Carcinoembryonic antigen (CEA), neuron‐specific enolase (NSE), and the BB isoenzyme of creatine kinase (CK‐BB) were evaluated before therapy in the sera of 195 patients with histologically confirmed small cell lung cancer (SCLC) in a prospective multicenter trial. Forty‐four percent (84 of 193) of all patients had CEA levels higher than 5 ng/ml, 66% (111 of 168) had NSE levels higher than 12.5 ng/ml, and 32% (40 of 123) had CK‐BB levels higher than 10 ng/ml. Clear pathologic levels were less frequently observed. Significantly higher pretreatment titers for CEA, NSE, and CK‐BB were found in patients with bone marrow and/or liver metastases. The most elevated marker levels were observed in the group of nonresponding patients with bone marrow and/or liver metastases. Only a slight correlation between the pretreatment CEA level and survival time could be observed. Patients with pathologic NSE (greater than or equal to 30 ng/ml) levels and, in particular, those with pathologic CK‐BB (greater than or equal to 25 ng/ml) levels had a significantly shorter median survival than those with normal or elevated levels. In addition, a positive linear correlation between pretreatment NSE and CK‐BB (n = 116, r = 0.54) levels was found, but CEA levels did not correlate with other marker levels. From these data it is concluded that pretreatment CEA, NSE, and CK‐BB levels are helpful in the clinical management of a subset of patients with SCLC, i.e. those with bone marrow and/or liver metastases.

Evaluation of serum neural cell adhesion molecule as a new tumor marker in small cell lung cancer

Background. Small cell lung cancer (SCLC) is distinguished from other histologic types of lung cancer by possessing a variety of neuroendocrine properties. Neuron‐specific enolase (NSE) is the most frequently elevated tumor marker for patients with SCLC at diagnosis. To assess the value of neural cell adhesion molecules (NCAM), another possible tumor marker for small cell lung cancer, NCAM was evaluated in the sera of patients with histologically confirmed SCLC in two prospective multicenter trials.

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

SummarySmall-cell lung cancer (SCLC) and non-smallcell 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 (α-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.

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When [125I]IGF-I or [125I]IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. The dissociation constants were determined to be 0.106 nM for IGF-I and 0.209 nM for IGF-II binding. Cross-linking of [125I]IGF-I or [125I]IGF-II to the CMs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range 24-32 kDa. The binding was competitively inhibited by addition of cold IGF-I and IGF-II but not by insulin. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.

Molecular Cloning of IGFBP-5 from SCLC Cell Lines and Expression of IGFBP-4, IGFBP-5 and IGFBP-6 in Lung Cancer Cell Lines and Primary Tumours

We showed recently that insulin-like growth factor binding protein (IGFBP)-1, -2 and -3 are differentially expressed in lung cancer and permanent lung cancer cell lines. Elevated levels of IGF binding capacity in serum of lung cancer patients were also reported. The function and tissue specificity of IGFBP are still obscure but they are probably local regulators of IGF action. Here we show the expression of IGFBP-4 transcripts in 11/11 small cell lung cancer (SCLC) cell lines, in nine out of 11 non-small cell lung cancer (NSCLC) cell lines, in 11/11 lung tumour specimens (10 derived from patients with NSCLC and one from SCLC origin) and in normal lung. In addition we isolated IGFBP-5 cDNA from lambda gt10 libraries of SCLC cell lines. With this IGFBP-5 cDNA we detected transcripts of different lengths in seven out of 11 SCLC cell lines, in 11/11 lung cancer specimens but only in one out of 11 NSCLC cell lines and in normal lung. IGFBP-6 was not detected in northern analysis of any tested SCLC cell line but it was expressed in nine out of 11 NSCLC cell lines and in nine out of 11 human lung cancer specimens and in normal lung.

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN EXPRESSION IN HUMAN SMALL CELL LUNG CANCER CELL LINES

Insulin-like growth factor binding proteins (IGF-BP) are secreted by several human small cell lung cancer cell lines (SCLC). In order to identify the IGF-BPs from SCLC cell lines the RNA from 10 different SCLC cell lines was analyzed by Northern blot analysis with the probes for three different IGF-BPs, IGFBP-1, IGFBP-2, and IGFBP-3. No hybridization signal could be detected with the probes encoding for IGFBP-1 and IGFBP-3. The hybridization with different IGFBP-2-specific oligodeoxynucleotide probes and with the corresponding full-length cDNA showed that all SCLC cell lines which secreted IGF-BPs express IGFBP-2.

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.