204 Biology I•'-• Use of tumor-specificpromoter for experimental gene therapy in small cell lung carcinoma N. Inase, Y. Yoshizawa. The Pulmonary Medicine, Tokyo Medical and Dental University, Tokyo, Japan Small cell lung carcinoma (SCLC) accounts for 15% to 20% of all lung cancers. Although SCLC is initially responsive to chemotherapy and radiotherapy, long-term survival is rare. As a suicide gene therapy, transduction of herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of ganciclovir (GCV) has been applied to many different types of malignant cells. Because HSV-TK gene should be expressed exclusively in tumorcells, tumor-specific promoters have been utilized to regulate the expression at the transcriptional level. Gastrin-releasing peptide (GRP) and neuron specific enolase (NSE) are known to be expressed in SCLC. In the present study, GRP and NSE promoters were applied to this system as a SCLC- specific promoter. Human SCLC cell lines (SBC3 and SBCS), a human lung adenocarcinoma cell line (A549), and a human uterine cervix epitheloid carcinoma cell line (Hela) were cultured. RT-PCR analysis demonstrated GRP expression in SBC5 cells and NSE expression in SBC3 cells. Luciferase activity of a reporting vector containing 1.2 kb- GRP promoter region (pGL2-GRP) was higher in SBC5 cells, and that of a reporting vector containing 1.1 kb-GRP promoter region (pGL2- NSE) was higher in SBC3 cells. After an expression vector containing GRP promoter-bound HSV-TK (pGRP-TK) or that containing NSE promoter-bound HSV-TK (pNSE-TK) were transfected into the cells, their sensitivity to GCV was measured by cell proliferation assay. After transfection of pGRP-TK, SBC5 cells became 100 times more sensitive to GCV, however, transfection of pNSE-TK did not affect the sensitivity of SBC3 cells to GCV. In nude mice, tumors of pGRP-TK transfected SBC5 regressed completely after intraperitoneal adminis- tration of GCV. GRP promoter might be a good tool for tumor-specific transduction of suicide genes in GRP-expressing SCLC. • Resting energy expenditure, body cell mass and the inflammatory response in patients with non-small cell lung cancer H. Scott, S. Davidson, D. McMillan, W. Watson, C. McArdie, R. Milroy. Stobhill Hospital, Glasgow, G21 3UW, UK Resting energy expenditure (REE) is known to be increased in non- small cell lung cancer (NSCLC) patients with weight loss (ref. 1). This increase is associated with the presence of an inflammatory response as indicated by the level of the acute phase protein re- sponse, measured as the C-reactive protein (CRP) (ref. 2). Such an inflammatory response has also been seen to occur before weight loss in patients with lung cancer (ref. 3), but it is not clear whether REE is also increased at this time. To examine these relationships 7 healthy male subjects and 12 patients with NSCLC and no significant weight loss underwent measurements of REE, total body potassium (TBK), C-reactive protein and albumin. REE was measured by indirect calorimetry. REE is conventionally adjusted for metabolically active tissue using total body water. However alterations in extracellular water components due to pathological states may confound these measurements, but the measurement of body cell mass derived from the largely intracellular TBK is not affected by these changes. TBK was measured using a scanning whole body counter. The cancer group had higher CRP (p < 0.001) and lower albumin concentrations (p < 0.05) compared with control. REE was approximately 15% higher in the cancer group. This study showed a significant relationship, in these weight stable patients with NSCLC, between the level of REE/body cell mass and their CRP concentrations (r = 0.753, p < 0.01). These results suggest that there is an association between the level of the inflammatory response and the increase in REE seen in patients with NSCLC, prior to onset of weight loss. References [1] Hansell DT, Davies JWL, Burns HJG. The effects on resting energy expenditure of different tumour types. Cancer 1986: 58:1739-1744 [2] Staal-van den Brekel A J, Dentener MA, Schols AMWJ, Buurman WA, Wouters EFM. increased resting energy expenditure and weight loss are related to a systemic inflammatory response in lung cancer patients. J Clin Oncol 1995: 13:2600-2605 [3] Scott HR, McMillan DC, CrUly A, McArdle CS, Milroy R. The rela- tionship between weight loss and interleukin-6 in non-small cell lung cancer. Br J Cancer 1996: 73: 1560-1562. 16• Overexpression of multidrug resistance-associated protein 3 in cisplatin-resistant cells established after a cycle of cisplatin-containing chemotherapy H. Kawai, K. Kiura, H. Ueoka, M. Tabata, I. Takata, N. Nogami, A. Hiraki, K. Chikamori, N. Horita, M. Hareda. Sec. Dept. of Med., Okayama Univ. Med. Sch., Okayama 700-8558, Japan We had established in vitro doxorubicin-, etoposide-, cisplatin-, and 7- ethyl-10-hydroxy-camptothecin (SN-38)-resistant lung cancer cell lines after continuous exposure of each drug for a long time and elucidated the mechanisms of drug resistance. It is a critical problem whether drug-resistance in these in vitro resistant cell lines reflects clinical drug-resistance. In this study, a pair of lung cancer cell lines were established from one patient with squamous cell carcinoma of the lung before and after a cycle of cispiatin-containing chemotherapy. EBC-2/R cells established after chemotherapy were 2.2-fold more resistant than the EBC-2 cells established before chemotherapy to cisplatinin termsof concentration of 50% inhibition (IC50) values by a 3-[4,5-dimethyl-thizol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) assay. Using this cisplatin resistant cell line established in vivo, the mechanismsof cisplatin-resistance were analyzed. EBC-2/R cells showed a substantial resistance to cisplatin, a decrease in intracellular accumulation of cisplatin, an increase in intracellular concentration of glutathione (GSH), and overexpression of multidrug resistance- associated protein (MRP) 3 when compared to EBC-2 cells. A cycle of chemotherapy could not affect other drug resistant mechanisms such as multidrug-resistance (MDR)-l/P-glycoprotein, MRPs 1, 2, 4, 5, lung resistance-related protein (LRP), metallothionein Ila (MT-Ila), glutathione S-transferase-pi (GST-pi), gamma-glutamylcysteine syn- thetase (gamma-GCS) (light & heavy chain), and excision repair cross complementing 1 (ERCC1). Both MRP3 and GSH were selected or induced at an early course of chemotherapy, and might be related to the clinical cisplatin-resistance. I - ~ - ~ The role of upstream stimulatory factor in expression of the vasopressin gene in lung cancer J.M. Coulson, J.L. Edgson, R.J. Mulgrew, J.P. Quinn, P.J. Woll. University of Nottingham, Nottingham, ElK The neuroendocrine phenotype distinguishes small cell lung cancer (SCLC) from the majority of other normal or neoplastic lung cell types; identification of transcription factors which regulate expressio of neuropeptides will aid in the design of novel molecular therapies. Arginine vasopressin can act as an autocrine growth factor in SCLC and we have characterised the 5 ~ vasopressin proximalpromoter, which directs gene expression specifically to this tumour type. The restricted expression relies on both a repressor and an enhancer, which includes an E-box (A) transcription factor-binding motif. This bound a heterodimer of upstream stimulatory factor-1 (USF-1)/USF- 2 in electrophoretic mobility shift analysis (EMSA) (Coulson et al, Biochem J 344; 961-70, 1999). RT-PCR and EMSA binding, in both SCLC and other cell lines with undetectable vasopressin expression, demonstrated USF factors. However, E-box A was most strongly bound by USF in a SCLC cell line with very high-level vasopressin expression. We now report that co-transfection of USF with vasopressin promoter- driven reporter constructs not only increased reporter gene expression in SCLC, but also switched on the promoter in non-SCLC, where it is normally inactive. This USF activation in non-SCLC was reduced by expression of the repressor, which normally restricts vasopressin ex- pression. Although USF activation in non-SCLC was partly via E-box A, we have shown by site-directed mutagenesis that this is not the major