Levels of Complement Regulatory Molecules in Lung Cancer: Disappearance of the D17 Epitope of CD55 in Small-cell Carcinoma

The levels of complement-regulatory molecules (complement receptor type one [CR1], decay-accelerating factor [DAF], membrane cofactor protein [MCP], and an inhibitor of membrane attack complex [CD59]) in lung cancer cells were analyzed to investigate the relation between their expression and histological subtypes, and the possibility of homologous complement deposition on cancer cells. In 25 cell lines (10 adenocarcinoma, 3 large-cell carcinoma, 7 small-cell lung cancer [SCLC], and 5 squamous cell carcinoma), flow cytometric analysis revealed that MCP was expressed in all cell lines, whereas none of the cell lines was CR1-positive. CD59 was detected in all cells. The DAF epitope defined by IA10 was expressed in all cells except one large cell carcinoma cell line. However, another epitope for anti-DAF monoclonal antibody, D17, was not detected in 5 (71.4%) SCLC and in 4 (22.2%) non-small-cell lung cancer. This disparity was seen in most cell lines, irrespective of histological subtypes. The loss of D17 reactivity seemed to be pertinent to malignant phenotype, because most of the normal pulmonary cells possessed the D17 epitope. Furthermore, a cell line lacking DAF (IA10⁻/D17⁻) allowed alternative pathway-mediated homologous complement (C3) deposition after pretreatment with anti-MCP antibody. This raises a new possibility for immuno-targeting of cancer. These cell lines should be useful in studying the biology of lung cancer.     

Gene expression profiles in human non-small and small-cell lung cancers

Suppression subtractive hybridisation (SSH) was performed comparing normal bronchial epithelial cells with a lung squamous cell carcinoma (SCC) and a metastatic small-cell lung carcinoma (SCLC). The sequence analysis of four cDNA libraries revealed 869 individual sequences. Of these, 342 were tested using northern blots of lung cancer cell lines representing the three major subtypes (SCC, adenocarcinoma, SCLC) which confirmed the differential expression of 236 cDNAs. The extended analysis of 31 randomly chosen fragments confirmed the validity of the approach to identify genes associated with lung cancer development. Additionally, five novel full-length cDNA were isolated encoding the microtubule-associated proteins 1A/1B light chain 3, the epithelial V-like antigen 1 (EVA1), the GTP-binding protein SAR1, a new member of the S100-type calcium binding protein family and a new homeobox-containing gene.     

肺癌细胞株中GEM和CDDP药物敏感性相关基因的研究

背景与目的 筛选小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞株中与健择(gemcitabine hydrochloride,GEM)和顺铂(cisplatin,CDDP)药物敏感性相关的基因,有助于进一步阐明抗癌药物作用机制,为克服抗癌药物的耐药性、研制开发新的抗癌药物提供新线索,同时也将为临床的个体化治疗提供理论依据.方法 采用MTT比色分析法测定4株SCLC细胞株和6株NSCLC细胞株对CDDP和GEM的药物敏感性,同时应用cDNA macroarray技术检测10株肺癌细胞株中1 291个药物敏感性相关基因的表达状态,分析二者之间的相关性.结果 与GEM药物敏感性呈明显正相关(r≥0.632,P<0.05)的基因有6个;与CDDP药物敏感性关联的基因共有45个;与GEM、CDDP敏感性关联(r≥±0.4)的基因有41个;肺癌细胞系中与GEM和CDDP两类药物敏感性呈明显相关的基因足Metallothinein(信号转导分子)、Cathepsin B(组织蛋白酶B)和TIMP1(生长因子);肺癌细胞系中与GEM、CDDP药物敏感性相关联的基因主要分布于Metallothinein、CathepsinB、牛长因子TIMP1等类别.结论 SCLC和NSCLC细胞株中GEM、CDDP存在药物敏感性相关基因,其中Metallothinein、Cathepsin B和TIMP1基因与GEM药物敏感性呈正相关,与CDDP药物敏感性呈负相关.     

Recent Advances and Future Strategies for Immune-Checkpoint Inhibition in Small-Cell Lung Cancer

Small-cell lung cancer (SCLC) is distinguished from non–small-cell lung cancer by its rapid growth and more frequent metastases. Although patients with SCLC are highly responsive to chemotherapy and radiation therapy, long-term prognosis remains poor, with relapse and disease recurrence occurring in almost all cases. Whereas combination chemotherapies continue to be the standard of care in extensive-stage SCLC, there is value in exploring whether immune-checkpoint inhibition is an effective treatment strategy, given the durable responses in non–small-cell lung cancer. Data from SCLC trials have shown clinical activity and response to cytotoxic T-lymphocyte antigen-4 protein and programmed cell death-1 blockade, suggesting that antibodies targeting these pathways may be effective in improving survival outcome. However, data on clinical activity by programmed cell death-1 ligand expression in SCLC are not widely available. Limited data indicate that programmed cell death-1 ligand expression may not be an ideal biomarker for patient selection. Continued research is necessary to better optimize patient selection and response to therapy.     

Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.

We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12, 13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras codon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extra-pulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61 respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SCLC.     

Androgen receptors, androgen-dependent proliferation, and 5 alpha-reductase activity of small-cell lung cancer cell lines

We investigated the influence of testosterone on binding to established small-cell lung cancer (SCLC) cell lines and were able to show specific high-affinity and low-capacity binding sites in some cell lines with a typical receptor size, using sucrose density gradient centrifugation. In addition, we demonstrated marked growth stimulation with testosterone and dehydrotestosterone using different androgen-receptor-positive small-cell lung cancer cell lines. This growth stimulation could be counteracted by the addition of anti-androgens like cyproterone acetate or flutamide. The presence of 5 alpha-reductase activity, 17 beta-hydroxysteroid-dehydrogenase and 3 alpha-hydroxysteroid-dehydrogenase activities could be demonstrated, suggesting testosterone metabolism of small-cell lung cancer cell lines.     

Characterization of four new cell lines derived from small-cell gastrointestinal carcinoma

Four human small-cell gastrointestinal carcinoma cell lines were established from tumor tissues of patients with esopha-geal, gastric or rectal cancer, and were studied morphologically and biochemically in comparison with small-cell lung carcinoma (SCLC) cell lines and common gastric cancer cell lines. Cells from all the small-cell gastrointestinal carcinoma lines were as small as classic SCLC cells and had characteristic neurosecre-tory granules. Cells from only one line grew as tightly packed spherical aggregates of floating cells, and those of the other 3 grew attached to substrate. Although high levels of creatine kinase brain isoenzyme (CK-BB) were detected in all 4 cell lines, 2 of them showed low levels of aromatic L-amino-acid decarbox-ylase and 3 had low levels of neuron-specific enolase (NSE). None of the lines showed simultaneous elevation of enzymes. C-myc, N-myc, and L-myc were not amplified in any of the cell lines, but c-myc mRNA was expressed in 2 lines. Our findings indicate that all small-cell gastrointestinal carcinoma cells examined belong to the variant type which is used in the classification of SCLC. Furthermore, the ECC18 line, derived from esopha-geal cancer, seemed to be of true endocrine cell origin, while the 3 other small-cell gastrointestinal carcinoma lines seemed to arise via neoplastic neometaplasia from adenocarcinoma cells to endocrine cells.     

肺癌细胞株中顺铂药物敏感性的相关基因

目的 筛选小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)细胞株中与顺铂(DDP)药物敏感性相关的基因.方法 采用二苯基溴化四氮唑蓝(MTT)比色分析法,测定4株SCLC细胞株和6株NSCLC细胞株对DDP的药物敏感性;应用cDNA微阵列技术检测肺癌细胞株中1291个药物敏感性相关基因的表达状态,并分析基因表达与DDP敏感性之间的相关性.结果 Metallothionein、Cathepsin B、TIMP1、TNF-R1、TGFβ-induced 68000、Cathepsin L、Galectin-1、Annexin 11、PAI-1、IGFBP4、UPAR、Jagged、CD13、α1 A-AR、EphA2(Eek)、APC、RhoC、Fibromodulin、GATA-6、HSC 70共20个基因,在SCLC和NSCLC细胞株中均与DDP的药物敏感性呈负相关,只有Procoagula和MDM2与DDP的敏感性呈正相关.VHL、MMP-7、Elongin A、GSK-3β、SLC、Galectin-3、Integrinβ5、Moesin、IKKβ和ETV 1等10个基因,只在SCLC细胞株中与DDP的药物敏感性呈负相关,而AT2与DDP敏感性呈正相关.Clusterin、FG FR-2、Thrombospondin 1、HSP 32、Lactate dehydrogenase A、P300、Thymosinβ10、CD81、C/EBPγ和Rak等10个基因,只在NSCLC细胞株中与DDP的药物敏感性呈负相关,而只有CaMKK和TPA与DDP的敏感性呈正相关.结论 与DDP药物敏感性相关的基因共有45个,其中共同表达于SCLC和NSCLC细胞株中的基因有22个,在SCLC细胞株中特异表达的基因有11个,在NSCLC细胞株中特异表达的基因有12个.     

Variable mutations of the RB gene in small-cell lung carcinoma

Loss of heterozygosity for chromosome 13q including the RB locus is a common genetic alteration in small-cell lung carcinoma (SCLC) as well as in retinoblastoma. We examined the RB cDNA sequences of exon 13 to 18 and exon 19 to 23 in 9 SCLC cell lines to detect mutations which cause inactivation of the remaining allele of the RB gene. Internal deletions of RB cDNA were observed in 3 of the 9 SCLC cell lines. In the Lu-24 cell line, a 114 base pairs (bp) deletion corresponding to exon 22 was due to abnormal splicing, which probably resulted from a two-base mutation within genomic exon 22, and aberrant 105 kilodaltons RB protein was detected by immunoprecipitation analysis. A base pair deletion within exon 20 in the Lu-135 cell line and a 1 bp deletion within exon 23 in the Lu-141 cell line were due to the deletions of the corresponding genomic DNA, and each deletion resulted in formation of a premature termination codon. These results indicate that both alleles of the RB genes are inactivated in SCLC by several different mechanisms, including small deletion, mutation and chromosomal loss.     

Specific sensitivity of small cell lung cancer cell lines to the snake venom toxin taipoxin

Small cell lung cancer (SCLC) is a malignant disease, for which no satisfactory treatment is presently available and consequently, new specific therapeutic targets are in high demand.

A global gene expression analysis previously performed, identified the neuronal pentraxin receptor (NPR) as highly and relatively specifically expressed in SCLC, consistent with the neuroendocrine features of this cancer. Normally, NPR is exclusively expressed in neurons, where it associates with the homologous proteins neuronal pentraxins 1 and 2 (NP1 and NP2) in complexes capable of binding the snake venom neurotoxin taipoxin.

The purpose of the present study was to assess the toxic effect of taipoxin in SCLC-cell lines and to determine if toxicity correlates to NPR and NP1 and NP2 expression levels.

NPR was detected by Western blot analysis in all the tested SCLC and in control cell lines of different origin. The receptor co-purified with cell membrane in SCLC, indicating that NPR is surface associated. Microarray signals for NP1 and NP2 mRNA was detected in a subset of SCLC-cell lines and validated by Northern blot analysis. Furthermore, NP1 protein was detected by Western blot analysis in a few SCLC-cell lines, but not in the control cell lines. A number of SCLC-cell lines showed marked sensitivity to taipoxin (IC₅₀: 3–130 nM) at toxin concentrations leaving the control cell lines unaffected. The sensitivity to taipoxin did not correlate with the expression levels of NP1 protein and NP2-mRNA, suggesting that expression of these proteins may not be required for taipoxin induced toxicity in SCLC.

The demonstrated toxic effect of taipoxin in SCLC may prove to be of importance for designing novel specific treatment modalities for this disease.     

Cancer vaccines for the treatment and prevention of non small-cell lung cancer

Cancer vaccines targeting non small-cell lung cancer (NSCLC) have been studied for decades; clinical trials, for the most part, have focused on the use of autologous and allogeneic whole-tumor cell vaccines. Recent advances in molecular biology and immunology, however, have allowed the identification of many tumor antigens involved in the generation of immunity to NSCLC. Although small-cell lung cancer (SCLC) is commonly thought of as an immunogenic tumor, it is now clear that NSCLC is also capable of eliciting an endogenous immune response in patients with the disease and, in fact, has a natural history that may make NSCLC more amenable to vaccine therapy as an adjuvant treatment strategy. This review will high-light the major components of the immune system that may potentially interact with tumor-associated proteins as well as outline the immunologic similarities and differences between SCLC and NSCLC. Tumor antigens that elicit immune responses in patients with NSCLC will be discussed. Finally, clinical trials of whole-tumor cell vaccines, both autologous and allogeneic, and tumor antigen-specific vaccines will also be discussed.     

Complementary DNA sequence encoding the major neural cell adhesion molecule isoform in a human small cell lung cancer cell line

The neural cell adhesion molecule (N-CAM), a member of the immunoglobulin gene super-family mediating homophilic cell-cell adhesion in a neuroendocrine system, is preferentially expressed in human small cell lung cancer (SCLC). Immunoprecipitation of a panel of SCLC cell lines by monoclonal antibodies (mAbs) specific for N-CAM detects mainly the 145-kDa isoform. This result was correlated with Northern blotting where a single 6.2-kb mRNA was detected in nine SCLC cell lines. To determine cDNA sequence encoding the N-CAM isoform, we selected several cDNA clones encoding N-CAM isolated from OS2-R, a SCLC cell line established in our laboratory. Based on the analysis of the full-length cDNA obtained from two clones, the sequence of this 145-kDa isoform was shown to be essentially identical to that of the 140-kDa N-CAM isoform of neuroblastoma except for a single base pair changed at position 1620 without changing amino acid encoded.     

In vitro response of human small-cell lung-cancer cell lines to chemotherapeutic drugs; no correlation with clinical data.

Three cell lines derived from small-cell lung carcinoma (SCLC) tumors of patients who had no clinical response after treatment with a multi-drug regimen were compared to 3 cell lines derived from tumors of patients who, upon treatment, showed a complete clinical response. These 2 groups of cell lines were considered to represent the in vitro counterparts of the 2 extremes of the clinical spectrum of sensitivity for chemotherapeutic drugs in small-cell lung cancer. To assess whether the in vivo (in)sensitivity of a tumor to a certain drug regimen is retained in vitro, the cell lines were tested for drug sensitivity using the microtiter-well tetrazolium assay and the results were compared with the in vivo data. No correlation was found. Since in vitro models using cell lines are based on the assumption that a cell line reflects the properties of the tumor from which it is derived, several additional parameters such as MAb staining against different SCLC-associated antigens and DNA content were analyzed in the biopsies and the cell lines. The results showed that selection of discrete tumor-cell populations in vitro occurs. Results of in vitro chemosensitivity testing for individual SCLC patients should be interpreted with caution.     

High frequency of somatically acquired p53 mutations in small-cell lung cancer cell lines and tumors.

We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Abnormalities of p53 were found in 16/16 cell lines (100%) and in 16/20 tumors (80%). In the SCLC cell lines, mutations (59% missense, 18% nonsense and 23% splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these data that: (1) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events.