SummaryAs a first step towards elucidating the potential role(s) of bcl-2 and bcl-2-related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-xl-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl-2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts.

Summary The present study examines the relationship between neuroendocrine (NE) differentiation and the clinical behaviour of non-small cell lung cancer (NSCLC). Retrospective (n = 315) and prospective (n = 44) cohorts of non-small cell tumours were obtained from surgically treated cases of lung cancer, comprising 218 squamous cell carcinomas, 65 adenocarcinomas, 51 adenosquamous carcinomas, and 25 large cell undifferentiated carcinomas. Paraffin wax embedded and fresh frozen tissue sections were stained for the NE markers neurone specific enolase, creatine kinase-BB, bombesin, neurotensin, chromogranin A, synaptophysin and UJ-13A. The expression of two or more markers was observed in 30% of cases, and was taken to identify NE-NSCLC. A statistically significant correlation between nodal status and NE differentiation (P = 0.05), and disease stage and NE differentiation (P = 0.04) was observed. However, there was no correlation between NE differentiation and survival. These findings suggest that NE-NSCLC, analogous to SCLC is more highly metastatic than non-NE-NSCLC.

Summary The production of insulin-like growth factor I (IGF-I) and IGF-I binding proteins (BPs) by human lung tumour cell lines in vitro has been examined and the levels of these substances in the serum of lung cancer patients investigated. While small cell lung cancer (SCLC) cell lines secreted both IGF-I and BPs, non-small cell lung cancer (NSCLC) cell lines secreted BPs only. No evidence of increased serum IGF-I levels was obtained in a cohort of 52 lung cancer patients having SCLC and NSCLC histologies. In contrast, serum levels of low molecular weight BPs were markedly elevated in the majority of lung cancer patients.The detection of elevated immunoreactive insulin-like growth factor-I (IGF-I) in human lung tumours (Minuto et al., 1986;Macaulay et al., 1988a) Healthy adult male and female non-smokers (n = 32), and male and female normal smokers (n = 31) were included in the study as controls. The age range for controls was 23-81 years and for lung cancer patients 39-79 years.Pre-treatment serum samples were prepared immediately after collection and stored at -70'C before assay. IGF-I determinationsA radioimmunoassay (RIA) kit (Amersham International, Aylesbury, UK) and a somatomedin C (SM-C) immunoradiometric (IRMA) kit (Immunodiagnostics Ltd, Tyne and Wear, UK) were used for the quantitative measurement of IGF-I in conditioned media and serum samples. Recombinant human IGF-I was used as standards in both assays. The rabbit antiserum used in the competitive RIA was raised against a recombinant analogue of IGF-I, shows 100% cross-reactivity with human IGF-I and 0.5% crossreactivity with human IGF-II. Fifty per cent displacement of tracer occurs with insulin at 2,000 ftunits ml-' (normal range of insulin in fasting individuals is 4-30 gunits ml-' in serum). Phase separation was achieved using Amerlex-M donkey anti-rabbit reagent (Amersham UK). Assay sensitivity is 100pg per tube.The non-competitive SM-C IRMA employed a two site immunoradiometric assay. Briefly standards/samples were incubated simultaneously with a mouse monoclonal anti-SM-C lgG immobilised on the inside walls of test tubes and a '25I-labelled mouse monoclonal antibody directed against a second IGF-I epitope. Unbound materials were then removed by decanting and washing the tubes. The antisera show 3% cross-reactivity with IGF-II and did not cross-react at all with insulin or pro-insulin. The lowest detectable level of SM-C that could be distinguished from the zero standard was 8 mU ml' at the 95% confidence limit.To separate binding protein from IGF-I, serum samples and cell conditioned media were extracted by incubation in 50 of acid extraction solution (supplied by Immunodiagnostics Ltd) for 10min at room temperature. Neutralising solution (500pl) (supplied by Immunodiagnostics Ltd) was then added to each sample. This extraction procedure yields aproximately 100% recovery of SM-C in patient samples. The neutralised, extracted conditioned media and serum samples, and unextracted serum samples were then used in the Amersham RIA and the IRMA.

The secretion of insulin-like growth-factor-binding proteins (IGFBPs) and expression of the genes encoding IGFBP-1, IGFBP-2 and IGFBP-3 have been studied in a panel of cell lines derived from breast carcinomas, Wilms' tumour, neuroblastoma, retinoblastoma, colon carcinoma, liver adenocarcinoma, Burkitt's lymphoma and a non-small-cell lung carcinoma. All cell lines, with the exception of the Burkitt's lymphoma cell line, secreted IGFBPs, as detected by affinity labelling. A 34-kDa BP was present in the conditioned media of all IGFBP-secreting cell lines, whereas BPs ranging from 18 kDa to 53 kDa were variably secreted. All IGFBP-secreting cell lines expressed the IGFBP-2 gene as determined by Northern blot analysis. The Wilms' tumour, the neuroblastoma and the retinoblastoma cell line expressed the IGFBP-2 gene only. All other cell lines, with the exception of the Burkitt's lymphoma, expressed the IGFBP-2 gene and, in addition, either the IGFBP-1 gene and/or the IGFBP-3 gene. IGFBP-1 gene expression could be detected by reverse transcriptase polymerase chain reaction only. IGFBP-3 gene expression was detected by Northern blot analysis, but transcripts were less abundant than IGFBP-2 mRNAs. These findings indicate that the expression of multiple BP genes and the secretion of BPs may be a common property of tumour cells.

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