Julie G. Reeve

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.

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.

Non-P-glycoprotein-mediated multidrug resistance with reduced EGF receptor expression in a human large cell lung cancer cell line.

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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.

Response to X-radiation and cytotoxic drugs of clonal subpopulations of different ploidy and metastatic potential isolated from RIF-1 mouse sarcoma.

Clonal subpopulations of different ploidy values and metastatic capacities, isolated from the RIF-1 mouse sarcoma, have been tested for in vitro X-radiation sensitivity, for in vitro sensitivity to adriamycin and for in vitro and in vivo sensitivity to melphalan and CCNU. Following X-radiation, no consistent differences in the survival curve characteristics (Do and n) of diploid, tetraploid and octoploid cells were observed. In addition no relationship between radiation response and metastatic capacity was observed. For drug response, no marked differences were found in the dose response curves of RIF-1 clones treated in vitro with adriamycin. However, a wide variation in the responses of RIF-1 clones to in vitro melphalan treatment was observed which was independent of both ploidy and metastatic capacity. Although the responses of RIF-1 clones to in vitro CCNU treatment were similarly independent of metastatic capacity, a clear relationship between CCNU sensitivity and ploidy was observed. Thus, all diploid RIF-1 clones were markedly more sensitive to CCNU treatment than either tetraploid or octoploid RIF-1 clones. For both melphalan and CCNU treatment the relative sensitivities in vitro correlated with in vivo sensitivities as assayed by clonogenic cell survival.

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.

Chapter 21 Expression of monoclonal antibody-defined lung tumour antigens in drug resistant lung tumour cell lines

Abstract The expression of monoclonal antibody (Moab)-defined lung tumour antigens has been investigated in in vitro -derived multidrug-resistant (MDR) variants of small cell (SCLC) and non-small cell (NSCLC) lung tumour cell lines. The majority of Moabs tested reacted equally well with MDR and drug-sensitive SCLC and NSCLC cells. However, in contrast to drug sensitive SCLC cells, MDR-SCLC cells failed to express the glycolipid antigen defined by Moab LCA1/LC38, showed increased expression of 2 glycoproteins, defined by Moabs LAM8 and SWA 4, and increased expression of the sugar epitope defined by Moab MOv15. Drug resistant large cell lung cancer cells also showed increased expression of the glycoprotein defined by Moab SWA 4 compared to the parent line which failed to react with this Moab. No significant differences were observed in the expression of lung tumour antigens by drug sensitive and resistant adenocarcinoma cells.

Chapter 13 Identification of antigenic phenotypes characterising lung tumour cell lines in vitro

Abstract The relationships between classic and variant small cell lung cancer (SCLC) cell lines and between variant SCLC (SCLC-V) and non SCLC (NSCLC) cell lines has been explored using a panel of selected monoclonal antibodies (Moabs). The aims of this study were to identity cell surface antigenic phenotypes which characterise the various lung tumour types and to relate the expression of lung tumour antigens to differentiation status. These studies have identified antigenic phenotypes that (1) distinguish classic from variant SCLC cell lines; (2) distinguish SCLC (irrespective of subtype) from NSCLC; (3) correlate with the expression of L-dopa decarboxylase, neuron specific enolase and creatine kinase BB.

Evidence that multidrug resistance in Chinese hamster ovary cells is associated with alterations in the endoplasmic reticulum

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