Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.     

Interaction of recombinant human tumor necrosis factor and etoposide in human lung cancer cell lines

Studies have suggested that recombinant tumor necrosis factor-alpha (TNF-alpha) may potentiate the killing of murine tumor cells by drugs targeted at DNA topoisomerase II. We have examined the combined cytotoxic effects of the topoisomerase-targeted drug etoposide and TNF in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines using clonogenic assays and a novel flow cytometry technique relying on differential uptake of fluorescein diacetate (FDA) and propidium iodide (PI) by viable and nonviable cells. Good correlation of IC50 determinations for etoposide were noted between clonogenic assays and the FDA/PI technique for both classic and variant SCLC cell lines. The effects of etoposide on the classic SCLC line H209 were potentiated by TNF with a decrease in the IC50 from 3.3 microM to 1.0 microM as determined by FDA/PI. Tumor necrosis factor alone had little effect on the growth or cloning efficiency of H209 cells. Tumor necrosis factor alone stimulated the growth and cloning of variant SCLC line N417, but the cytotoxicity of etoposide was not potentiated by TNF in N417 cells. Tumor necrosis factor alone inhibited the growth and cloning of the NSCLC line H125 but exerted a marked protective effect against higher concentrations of etoposide. It appears that the interaction of TNF with etoposide varies between cell lines and between subclasses of human lung cancer.     

Oncogene amplification and chromosomal abnormalities in small cell lung cancer

Twelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto-oncogene family (c-myc, N-myc, and L-myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto-oncogene family. Amplification of c-myc was observed in only one small cell lung line--a "morphological variant". One "classic" small cell lung cancer line expressed c-myc but had no obvious amplification of the gene. N-myc and L-myc were more commonly amplified than c-myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to "classic" or "variant" characteristics, it was found that the "classics" had deletions of the short arm of chromosome 3, whereas the "biochemical variants" had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23-3p24.     

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, 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.     

Multiple neuropeptides mobilise calcium in small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin, galanin and neurotensin

The neuropeptides vasopressin, bradykinin, cholecystokinin, galanin, neurotensin and gastrin-releasing peptide stimulate rapid, transient increases in cytosolic Ca2+ in small cell lung cancer cell lines at nanomolar concentrations. Responsiveness to individual peptides is heterogeneous among the diverse cell lines, but the ability to respond to regulatory peptides is a general phenomenon. Peptide responses demonstrate homologous desensitisation and are blocked by ligand-specific antagonists, indicating that they are mediated by distinct receptors. Many neuropeptides are also secreted by small cell lung cancer. Here we suggest that multiple autocrine and paracrine interactions regulate its growth.     

Amplification of the N-myc oncogene in an adenocarcinoma of the lung

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.     

NCAM: a surface marker for human small cell lung cancer cells

Immunocytochemical and immunochemical techniques were used to study the expression of the neural cell adhesion molecule (NCAM) by human lung cancer cell lines. Intense surface staining for NCAM was found at light and electron microscopic levels on small cell lung cancer cells. The NCAM polypeptide of Mr 140,000 (NCAM 140) was detected by immunoblotting in all of 7 small cell lung cancer cell lines examined and in one out of two of the closely related large cell cancer cell lines: it was not detected in cell lines obtained from one patient with a mesothelioma, in two cases of adenocarcinoma, nor in two cases of squamous cell cancer. In contrast, neuron-specific enolase was found by immunoblotting in all the lung cancer cell lines tested and synaptophysin in all but the adenocarcinoma cell lines. These antigens were localized intracellularly. The specific expression of NCAM 140 by human small and large cell lung carcinomas suggests its potential as a diagnostic marker.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Very Long-Chain Acyl-CoA Synthetase 3: Overexpression and Growth Dependence in Lung Cancer

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65–76%, and the ability of tumor cells to form colonies in soft agar suspension by 65–80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer.     

The role of PKCalpha and RLIP76 in transport-mediated doxorubicin-resistance in lung cancer

In deletion mutant analyses of potential phosphorylation sites in RLIP76, we identified T297 and S509 as targets for phosphorylation by PKCalpha. Phosphorylation at T297 increased doxorubicin (DOX)-transport activity approximately 2-fold for RLIP76 purified from recombinant source, or from three small (H69, H1417, H1618) and three non-small cell, one each derived from H226 (squamous), H358 (bronchio alveolar), and H1395 (adenocarcinoma) lung cancer cell lines. T297 phosphorylation conferred sensitivity to tryptic digestion at R293. The specific activity for DOX-transport by RLIP76 purified from non-small cell, which was primarily in the phosphorylated form, was approximately twice that in small cell lung cancer cell lines. These finding offer a novel explanation for the observed intrinsic differences in sensitivity to DOX between non-small cell and small cell lung cancer cell lines.     

牛至油体外抗肿瘤活性研究

目的观察牛至油对肿瘤细胞株的生长抑制作用。方法采用MTT法检测不同浓度的牛至油对体外培养的多种肿瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。结果不同浓度牛至油作用后,人肝癌细胞系HepG2、人子宫颈癌细胞系JTC-26和肺癌细胞系A549出现增殖阻滞。MTT法确定牛至油对肝癌HepG2的IC50为118μg/ml;人子宫颈癌JTC-26的IC50为118μg/ml;肺癌A549的IC50为59μg/ml。结论牛至油具有体外抗肿瘤活性。     

Quantitative description of classic and variant small cell lung cancer cell lines by nuclear image cytometry

Six small cell lung cancer (SCLC) cell lines were examined using nuclear image analysis to find features characteristic of the classic and the variant type of SCLC. On the basis of their biochemical and biological properties three of these cell lines have been shown to represent the classic types, and three represent the variant type of SCLC. Using a combination of the image-derived run length, density, and geometric features, it was possible to distinguish between the classic and variant SCLC cell lines. The results of this study may be of help in assessing photometric features for the separation of the classic and variant subtypes of SCLC in solid tumors. Because of differences in treatment and prognosis between these two subtypes, such a separation may be of clinical value.     

Identification of Gene Expression Differences between Lymphangiogenic and Non-Lymphangiogenic Non-Small Cell Lung Cancer Cell Lines

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients.     

26S proteasome activity is down-regulated in lung cancer stem-like cells propagated in vitro

Cancer stem cells (CSCs) are a small subset of cancer cells capable of self-renewal and tumor maintenance. Eradicating cancer stem cells, the root of tumor origin and recurrence, has emerged as one promising approach to improve lung cancer survival. Cancer stem cells are reported to reside in the side population (SP) of cultured lung cancer cells. We report here the coexistence of a distinct population of non-SP (NSP) cells that have equivalent self-renewal capacity compared to SP cells in a lung tumor sphere assay. Compared with the corresponding cells in monolayer cultures, lung tumor spheres, formed from human non-small cell lung carcinoma cell lines A549 or H1299, showed marked morphologic differences and increased expression of the stem cell markers CD133 and OCT3/4. Lung tumor spheres also exhibited increased tumorigenic potential as only 10,000 lung tumor sphere cells were required to produce xenografts tumors in nude mice, whereas the same number of monolayer cells failed to induce tumors. We also demonstrate that lung tumor spheres showed decreased 26S proteasome activity compared to monolayer. By using the ZsGreen-cODC (C-terminal sequence that directs degradation of Ornithine Decarboxylase) reporter assay in NSCLC cell lines, only less than 1% monolayer cultures were ZsGreen positive indicating low 26S proteasome, whereas lung tumor sphere showed increased numbers of ZsGreen-positive cells, suggesting the enrichment of CSCs in sphere cultures.     

The human homolog of the rodent immediate early response genes, PC4 and TIS7, resides in the lung cancer tumor suppressor gene region on chromosome 3p21

Recently, human chromosome band 3p21.3 was shown to undergo overlapping homozygous deletions in several small cell lung cancer lines further defining a putative tumor suppressor gene(s) region. We report the cloning and mutational analysis of a novel human gene, SKMc15, from the commonly homozygously deleted region in three small cell lung cancer lines (NCI-H1450, NCI-H740, GLC20). It has 11 exons ranging in size from 50 to 541 bp with an open reading frame of 442 amino acids. The gene covers 7 to 10 kb of genomic DNA; the message of 1.8 to 2 kb is expressed in all analyzed fetal and adult human and mouse tissues including heart, brain, placenta, lung liver, skeletal muscle, kidney, testis and pancreas and in small cell and non-small cell cancer lines. The intron/exon boundaries were used to analyze the gene for mutations by exon PCR-SSCP sequencing in 60 small cell lung cancer cell lines. No loss-of-function mutations were detected. The cDNA sequence has high homology, 75% at the protein level, to the rat early response gene PC4 and its murine homolog TIS7. In addition, the known partial sequence of the putative mouse interferon β2 (64 amino acids) gene is highly conserved in PC4/TIS7 (94%) and in SKMc15 (83%) at the amino acid level. The sequence TAAAT, which is thought to be involved in mRNA degradation, is present in the 3′ UTR of SKMc15 and in the 3′ UTR of PC4 and TIS7 genes. Received: 28 August 1996 / Revised: 18 October 1996     

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.     

Integrative genomics revealed RAI3 is a cell growth-promoting gene and a novel P53 transcriptional target

In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1-6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a > or =3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.