Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.

As 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-x1-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.

Insulin-like growth factor (IGF) and IGF binding proteins in growth hormone dysregulation and abnormal glucose tolerance in small cell lung cancer patients.

Growth hormone (GH) regulation, glucose tolerance and serum concentrations of insulin-like growth factor (IGF) and IGF binding proteins (IGFBP) have been investigated in small cell lung cancer (SCLC) patients. Elevated serum GH was observed in the patient and smoking control groups but not in non-smoking control subjects. Glucose suppression of GH was observed in the few SCLC patients with raised basal GH but most SCLC patients exhibited a paradoxical increase in GH following oral glucose. Abnormal glucose tolerance and insulin resistance with respect to plasma glucose was observed in most patients. Patients showing GH dysregulation exhibited higher serum concentrations of IGFBP-2 than those showing no increase in GH. Abnormal glucose tolerance was associated with decreased serum concentrations of IGF-I. Given reports of elevated IGFBP secretion in SCLC and inhibition of IGF-I bioactivity by IGFBPs, these findings may indicate that increased serum IGFBPs disrupt IGF-I regulation of GH secretion and glucose homeostasis.

Diminished expression of insulin-like growth factor (IGF) binding protein-5 and activation of IGF-I-mediated autocrine growth in simian virus 40-transformed human fibroblasts.

The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IG-FBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IG-FBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation.

[D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P induces apoptosis in lung cancer cell lines in vitro.

The broad spectrum antagonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P has been shown previously to inhibit the growth of small cell lung cancer cells both in vitro and in vivo. To elucidate further the pathways involved in the growth inhibitory actions of [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P we have examined the effect of this agent on cell viability and the induction of apoptosis in small cell and non-small cell lung cancer cells. Treatment of lung tumor cells with [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P caused a concentration-dependent loss of cell viability which was accompanied by the onset of apoptosis, as defined by cytological criteria and DNA fragmentation. This effect occurred in both small cell and non-small cell lung cancer cells and was not dependent on de novo protein synthesis. Such findings indicate that the antiproliferative action of [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P involves a signal transduction pathway for apoptosis.

Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors.

The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.

Insulin-like growth-factor-binding protein gene expression and protein production by human tumour cell lines.

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.

Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines.

BACKGROUND: The presence of multiple, low-molecular-weight, insulinlike growth factor (IGF)-binding proteins in lung tumor cell-conditioned medium and lung cancer patient serum has been recently reported. PURPOSE: To begin to elucidate the genetic basis for these observations, the present study examines the expression by lung tumor cell lines of three IGF-binding protein genes, namely, IGFBP-1, IGFBP-2, and IGFBP-3. Since IGF-binding proteins are thought to modulate the biologic action of the IGFs, the relationship between the expression of IGF-binding protein genes and the genes encoding IGF-I and IGF-II also has been investigated. METHODS: Gene expression was studied in four small-cell lung cancer (SCLC) and three non-small-cell lung cancer (NSCLC) cell lines using Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) for IGFBP-1. RESULTS: IGFBP-1 gene expression was detected by Northern blot analysis in one NSCLC cell line only. However, RT-PCR revealed that the IGFBP-1 gene was expressed in all four SCLC cell lines and in two of the three NSCLC lines. Northern blot analysis of IGFBP-2 gene expression demonstrated that all lung tumor cell lines expressed this gene. A low level of IGFBP-3 gene expression was detected in one SCLC cell line and in all three NSCLC cell lines. All lung tumor cell lines expressed the IGF-II gene as determined by Northern blot analysis. In marked contrast, none of the lines showed evidence of IGF-I gene expression using this method. However, RT-PCR revealed a low level of IGF-I gene expression in one SCLC and one NSCLC cell line only. CONCLUSIONS: These observations indicate 1) that IGF-binding proteins secreted by lung tumors are encoded by at least three different genes; 2) that there may be a close association between IGF-II and IGFBP-2 gene expression, such that, where there is production of IGF-II, IGFBP-2 is the principal BP; and 3) that the IGF-II gene is more widely expressed than the IGF-I gene in human lung tumor cell lines.

Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line.

The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors.

Flow cytometric and immunohistochemical analysis of p62c-myc oncoprotein in the bronchial epithelium of lung cancer patients.

The expression of p62c-myc in bronchial resection lines (BRLs) from lung cancer and control patients, has been examined by immunohistochemistry and parallel flow cytometry using antibodies directed against the p62c-myc oncoprotein. Both methods indicated a marked increase in nuclear p62c-myc levels in BRLs from tumour cases as compared to control BRLs. Immunohistochemistry also revealed greater cytoplasmic positivity in BRLs from cancer patients than from control cases. Flow cytometric quantitation of nuclear p62c-myc confirmed the immunohistochemical findings demonstrating that the median level of nuclear p62-myc fluorescence in BRLs from tumour cases was 1919 fluorescence units (FU) (range:216-7367 FU) and 144 FU (range:0-1365 FU) for non-tumour control BRLs. No consistent difference in p62c-myc fluorescence was observed between BRLs from smokers and non smokers. Both methods indicated that in lung tumour cases, nuclear p62c-myc was increased in histologically normal and abnormal BRLs, suggesting that hyperexpression of this protein is an early event preceding detectable morphological change. These results suggest that increased p62c-myc levels may be an early event in the pathogenesis of lung cancer.

Production of neuromedin B and neuromedin B gene expression in human lung tumor cell lines.

Gastrin-releasing peptide (GRP), a mammalian bombesin-like peptide, has been shown to be an important autocrine growth factor for small cell lung cancer (SCLC). However, not all SCLC cell lines express the GRP gene or respond mitogenically to GRP stimulation, suggesting the existence of other autocrine pathways in this tumor. Neuromedin B (NMB), the mammalian counterpart of amphibian ranatensin, has been shown to be a mitogen for SCLC cell lines in vitro. To determine whether NMB is a potential autocrine growth factor for lung tumors, NMB gene expression, peptide synthesis, and secretion have been investigated in a panel of SCLC and non-SCLC (NSCLC) cell lines. Northern blot analysis and enzymatic amplification from mRNA by polymerase chain reaction showed that the NMB gene was expressed in all SCLC and NSCLC cell lines examined. In contrast, the GRP gene was expressed in four of six classic SCLC cell lines but not in variant SCLC or NSCLC cell lines. Immunoreactive NMB was detected by radioimmunoassay in the majority of classic SCLC, in one of three variant SCLC and in one of three NSCLC cell lines, and secreted NMB was detected in medium conditioned by a SCLC and a NSCLC cell line. The present study also demonstrated the presence of immunoreactive GRP in the absence of detectable GRP gene expression. The antiserum used in the GRP radioimmunoassay failed to cross-react with NMB but showed some cross-reactivity with amphibian phyllolitorin raising the possibility that certain SCLC cell lines may produce a phyllolitorin-like peptide.

Neuroendocrine differentiation and clinical behaviour in non-small cell lung tumours.

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.

Mitogenesis and the search for new targets for anticancer drug design.

An exciting new approach to the treatment of cancer is the development of therapeutic strategies which target growth factors and the signal transduction pathways elicited by them. The rationale for targeting the processes which regulate cell proliferation rests on the contention that the malignant phenotype is maintained as a result of alterations in the biochemistry of growth control. The challenge is to design novel anticancer agents which exploit qualitative or quantitative differences in the biochemical elements controlling tumour cell growth and thereby achieve tumour selectivity. A wide variety of drugs are currently under development and include agents which block growth factor-receptor interaction, or which inhibit the action or formation of second messengers such as protein kinase C or phospholipase C. Although in its infancy, the use of inhibitors of growth factor action as antineoplastic agents has already proven effective against some tumours.

Chemosensitisation by verapamil and cyclosporin A in mouse tumour cells expressing different levels of P-glycoprotein and CP22 (sorcin).

The relationships between resistance to adriamycin, vincristine, colchicine and etopside, expression of P-glycoprotein and CP22 (sorcin), and resistance modification by verapamil and cyclosporin A have been studied in a panel of multidrug-resistant (MDR) mouse tumour cell lines. Whereas there was a generally good correlation between the degree of resistance and the amount of P-glycoprotein, no relationship between resistance and CP22 expression was seen. At 3.3 microM verapamil, the sensitisation of the MDR cell lines was no greater than that of the parent line. At 6.6 microM verapamil, however, sensitisation of the MDR lines generally exceeded that of the parent line, although the line CR 2.0, expressing very high levels of P-glycoprotein was an exception. Little sensitisation to etoposide was seen in any of the lines. When cyclosporin A was used as the sensitiser at either 2.1 or 4.2 microM, there was a greater effect in lines expressing moderate to high levels of P-glycoprotein than in the parent line, although this tendency was less for adriamycin than for the other cytotoxics. Sensitisation to etoposide was much greater with cyclosporin A than with verapamil. At low levels (less than 1 microM) of CsA, however, sensitisation to colchicine was greater in the parent line than in cell line CR 2.0. These studies indicate that chemosensitisation by verapamil and cyclosporin A is extremely complex, depending upon sensitiser dose, the particular cytotoxic and the cell line. At low doses of the sensitisers, the sensitisation may be greater in lines expressing low levels of P-glycoprotein than in lines showing high levels.

Production of immunoreactive insulin-like growth factor-I (IGF-I) and IGF-I binding proteins by human lung tumours.

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.

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69.

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.

Neuron specific enolase expression in carcinoma of the lung.

The value of neuron specific enolase (NSE) immunoreactivity as a marker for small cell lung cancer (SLC) has been assessed using a monoclonal antibody (MCAB) against NSE, MCAB specificity was confirmed using purified enolase isoenzymes, sections of human brain, a panel of lung tumours, neuroendocrine and non-neuroendocrine tumours and normal tissues. Using this MCAB in radioimmunoassay and immunohistochemistry, NSE immunoreactivity was detected in all SCLC material examined. However, considerable reactivity was also observed in a number of non-small cell lung cancer cell lines and tumour biopsy specimens. Furthermore, intratumoral heterogeneity with respect to NSE immunostaining was observed in several cases. Factors which may underlie such intratumoral phenotypic diversity were assessed using flow cytometry together with MCABs directed against both NSE and non-neuronal enolase. Such studies revealed that enolase expression in cells which were no longer actively proliferating differed markedly from that of cells in exponential growth. Furthermore, cells grown under conditions of increasing hypoxia exhibited increased enolase expression relative to those grown under oxygenated conditions. It is concluded from these studies that NSE immunoreactivity per se is an unreliable marker for the SCLC phenotype.