Cisplatin sensitivity and resistance in lung cancer

St3

Abstmcts

of CD24, called heat stable antigen, acts as a ligand for nmrine P-selectin. Latex beads coated with CD24 purified from carcinoma cell lines were shown to specifically bind to P-selectin as did activated platelets and a human lung adenocarcinoma cell line transfected with CD24. The CD24/P-selctin binding pathway could be important in the dissemination of small cell lung cancer by facilitating its interaction with endothelium [9]. To elucidate the molecular basis of the overexpression of CD24 in small cell lung cancer we analyzed the promoter activity of a 3.4 kb S-flanking region of the CD24 gene in transient luciferase reporter gene assays. The activity of the CD24 promoter was compared to SV40 promoter as positive control and to an inverted CD24 promoter as negative control in a series of lung cancer cell lines. Luciferase activity in the small cell lung cancer cell lines OH3, SW2 and H69 ranged between 0.8 and 1.5 of positive control. In contrast, no luciferase activity was seen in five non-small cell lung cancer cell lines. Subsequent testing S-truncations of the CD24 promoter allowed us to identify the core promoter fragment. A deletion fragment of 269 bp had maximal activity in small cell lung cancer cell lines (15- to 20-fold higher than control), while activity remained 2- to lo-fold below control in non-small cell lung cancer cell lines. We conclude that the CD24 promoter has a strong and cell-type specific activity and propose its exploitation to drive the expression of therapeutic genes in small cell lung cancer [lo]. References [l] De Leij I., et al. Int J Cancer 1994;Suppl8:60-63. [2] Vansant et al. Clin Nucl Med 1992;17:431-438. [3] Friisch BA, et al. Cancer Immunol Immunother 1996;24:55-63. [4] Zimmermann S, et al. Cancer Inununol Immunother 1997#:1-9. [5] Ziegler A, et al. J Nat1 Cancer Inst 1997;89:1027-1036. [6] Zangemeister-Wittke, Br J Cancer (in press). [7] Jackson D, et al. Cancer Res 1992;52:5264-5270. [8] Smith A, et al. Br J Cancer 1991;64:263-266. [9] Aigner S, et al. Blood 1997;89:3385-3395. [lo] Quintini G, et al. Proc Am Assoc Cancer Res 1998;39:520. Cisplatin sensitivity and resistance in lung cancer Saijo N, Nishio K, Kurokawa H, Tomonari A, Suzuki T. Pharmacology Diuision, National Cancer Center Research Institute and Medical Oncology Division, National Cancer Center Hospital, Tsukiji 5-l-1, Chuo-ku, Tokyo 104-0045, Japan. Cisplatin is one of the most active drugs and is most frequently used for the treatment of lung cancer. The mechanism of CDDP-resistance is extremely complicated. Intracellular accumulation, detoxification and DNA-repair influence the sensitivity or resistance to cisplatin. IC50 to CDDP and CDDP accumulation in 11 non-small cell lung cancer cell lines inversely correlated, suggesting that the accumulation is one of the most important mechanisms which determine cisplatin sensitivity. Glutathione is considered to be one of the factors

for cisplatin resistance because cisplatin resistant cells show increased GSH level and increased GSH content is reported in tumors resistant to CDDP. The question is whether increased GSH level really contributes to cisplatin resistance. rGCS (y glutamylcystein synthetase) is considered to be a rate limiting enzyme for glutathione synthesis. To evaluate the role of glutathione we transfected rGCS gene to human SCLC cells, SBC-3. rGCS transfectant was named as SBC3/rGCS. We evaluated intracellular GSH content, sensitivity to cisplatin, GSX pump activity and cellular platinum accumulation. SBC/yGCS (transfectant) overexpressed ?GCS but not MRP. Compared with parental SBC-3, increased intracellular GSH content and decreased sensitivity to cisplatin were observed in ?GCS transfectant. We next analyzed how cisplatin regulates rGCS expresion. The transcriptional activity of ?GCS promotor was analyzed by using deletion construct of rGCS gene. Deletion of the sequence from -1413 to -664 or -315 base pairs increased transcriptional activity. The deletion of the sequence from -241 to -192 bp also increased transcriptional activity. The transcriptional activity of rGCS gene in SBC3 cells was analyzed with or without exposure to cisplatin. Cisplatin at the concentration of 3 PM increased the transcriptional activity of rGCS gene to 246%. By using a similar method to the previous experiment, the proximal sequence - 192 to + 91 base pairs was demonstrated to be involved in cisplatin induced augmentation of transcriptional activity. &-acting DNA elements involved in CDDP induced up-regulation of the rGCS gene. The expression of rGCS gene was positvely or negatively regulated by the various part of promotors. Cisplatin-induced enhancement of ?GCS gene was regulated in the proximal GC rich regions of promotor. The regulation of this portion may contribute to the overcoming of cisplatin resistance. The effect of rGCS inhibitor, BSO was evaluated. The addition of BSO (10 PM) to transfectant induced a significant decrease of GSH content, however, GSX pump activity, Pt accumulation, and sensitivity to cisplatin, were not influenced. From these data it was suggested that another mechanism may contribute to cisplatin resistance. YCP, multidrug resistant protein (MRP), multicanalicular anion transporter (cMOAT) are physiologically known to export GSSG, leucotrien C4, cadmium-glutathione complex. The roles of these transporters are partly analyzed. Adriamycin-glutathione complex is demonstrated to be exported by MRP, and the transport of SN-38 is mediated by cMOAT. However, transporters for cisplatin are not clear yet. We tried to clone a new transporter from PC-14/CDDP and obtained SMRP (Short MRP) which was composed of 946 amino acids. SMRP had ABCs with walker A and B motifs as MRP, cMOAT and yeast YCF 1. So SMRP was considered to be a member of the ABC superfamily. This gene was mapped on chromosome 3 at band 27 by FISH analysis. MRP is mapped in chromosome 16. cMOAT is mapped in chromosome 10. So SMRP is determined to be a new member of the ABC superfamily. The tissue distribution of SMRP was analyzed. SMRT distributed in various organs as MRP. However, the expression in brains was significantly higher compared with that of MRP. In summary, SMRP is composed of 946 ammo

Abstracts

acids, it has two ABC regions with Walker motifs, it mapped on chromosome 3 at q27, it expressed in various tissues and was highly expressed in brain. The functional role of SMRP is under investigation in my laboratory. References

[l]

[2]

[3]

[4]

[5]

Morikage T, Ohmori T, Nishio K, Fujiwara Y, Takeda Y, Saijo N. Modulation of cisplatin sensitivity and accumulation by amphotericin B in cisplatin-resistant human lung cancer cell lines. Cancer Res 1993;53:3302-3307. Tomonari A, Nishio K, Kurokawa H, Arioka H, Ishida T, Fukumoto H, Fukuoka K, Nomoto T, Iwamoto Y, Heike Y, Itakura M, Saijo N. Identification of &-acting DNA elements of the human y-ghttamylcysteine synthetase heavy subunit gene. Biochem Biophys Res Communic 1997;232:522-527. Tmonari A, Nishio K, Kurokawa H, Fukumoto H, Fukuoka K, Iwamoto Y, Usuada J, Suzuki T, Itakura M, Saijo N. Proximal 5’-flanking sequence of the human y-glutamylcysteine synthetase heavy subunit gene is involved in cisplatin-induced transcriptional up-regulation in lung cancer cell line SBC-3. Biochem Biophys Res Commun 1997;236:616-621. Suzuki T, Nishio K, Sasaki H, Kurokawa H, Saito-ohara F, Ikeuchi T, Tanabe S, Terada M, Saijo N. cDNA cloning of a short type of multidrug resistance protein homologue, SMRP, from a human lung cancer cell line. Biochem Biophys Res Commun 1997;238:790-794. Kurokawa H, Nishio K, Ishida T, Arioka H, Fukuoka K, Nomoto T, Fukumoto H, Yokote H, Saijo N. Effect of glutathione depletion on cisplatin resistance in cancer cells transfected with the yglutamylcysteine synthetase gene. Jpn J Cancer Res 1997;88:108-110.

Radiotherapy

of small

cell lung cancer (SCLC) of Clinical Oncology, Western Trust, Edinburgh EH 4 2XlJ UK.

Gregor A. Department Hospital

NHS

General

Radiotherapy has provided the most significant improvement in survival of patients with SCLC achieved in the last decade. The chemosensitivity and often disseminated nature of SCLC made systemic chemotherapy the logical first line choice for therapy and clinical research. Unfortunately most of the currently available and tested chemotherapy strategies, such as dose intensification, achieved no major improvements in long term survival and over 90% of SCLC patients continue to relapse and die with chemoresistant and widespread tumour. Important prognostic factors associated with prolonged survival have been identified and allow patient categorisation at the time of diagnosis and first line induction therapy. For the ‘good prognosis’, group of patients (LTD, good PS, normal biochemistry, no significant weight loss and major response to induction chemotherapy) intrathoracic relapse and brain metastases are two common sites of failure. Thoracic irradiation (TI) and prophylactic cranial irradiation (PCI) have been known to halve the incidence of relapse at these sites for more

s9 than a decade. The lack of demonstrable survival benefit in individual studies as well as the legitimate concerns about increased short term toxicity of TI and reports of significant long term side effects associated with the use of PC1 have tempered enthusiasm for their use and lead to exclusion of PC1 from most of the formal multimodality protocols. Two events changed the scenario. The first was a series of overviews and a metanalysis [l] demonstrating that a 30% reduction of local intrathoracic failure led to 5.4% improvement in 3-year survival (8.9% vs. 14.3%). The second was a series of PC1 trials [2-41 and their metanalysis [5] which confirmed the ability of moderate doses of PC1 to significantly reduce brain metastases rates (HR 0.45) with no demonstrable increase in CNS toxicity. The metanalysis confirmed statistically the small, but consistently observed survival benefits of PCI. Both TI and PC1 are recommended as a standard treatment for the subgroup of patients with SCLC who have a realistic prospect of long-term survival. A number of factors need further research and clarification. They include: radiation dose and fractionation, timing and scheduling of radiation in relation to chemotherapy. For TI the issue of treatment volume also remains unresolved. Increasing radiation dose of TI and PC1 improves local control and may lead to survival advantage. In the case of TI this can be achieved by concurrent administration, but for PC1 toxicity concerns prevent this approach. Manipulation of fractionation may also provide a method of dose escalation by accelerating treatments or limiting long term toxicity. Hyperfractionated, accelerated schedules may achieve both. However the choice and method of administration of chemotherapy provides further variable and adds complexity to the design and execution of clinical trials testing these concepts. There are theoretical reasons and some clinical evidence suggesting early use of TI is more effective than delaying beyond 3-4 months from diagnosis. Further studies are necessary to substantiate this approach [6]. Concurrent chemoradiotherapy has gained widespread acceptance without a large body of convincing randomised clinical evidence of superiority. Phase II studies suggested a 4-year survival of 30%. A randomised intergroup trial comparing concurrent chemoradiotherapy with daily or hyperfractionated accelerated schedule has demonstrated a better local control in the accelerated arm with increased toxicity, but no significant survival benefit [8]. In many cases the clinical trial design is confounded and combines aspects of timing, fractionation and acceleration. Early results of JCOG trial suggest survival benefit for early concurrent administration [7]. Additional benefits can be achieved by judicial use of radiotherapy in patients with advanced and poor prognosis disease. In this setting cure is unattainable with currently available chemotherapy. Radiation offers useful, easy to administer and well tolerated palliation. In this setting we use low dose schedules with minimal side effects and low burden for the patient. TI and PC1 play an important role in the overall management of patients with SCLC. A number of questions relating to the choice of schedules and their optimal incorporation into multimodality protocols remain and will need close collaboration between medical and radiation oncologists