1. STUDIES OF INDUCIBILITY OF THE EXTRACELLULAR MATRIX PROTEIN CYR61 IN THE PROSTATE CARCINOMA CELL LINES LNCAP, PC-3 AND DU145 Ulrike Fiedler, *Uta Schmidt, # Christian Pilarsky, Claudia Eißrich and Manfred P. Wirth Klinik und Poliklinik für Urologie der TU Dresden, *Centre Medical Universitaire, Genf, # MetaGEN GmbH, Berlin INTRODUCTION The extracellular matrix signaling molecule Cyr61 has been first detected in serum-stimulated fibroblasts after serum starvation (O’Brien et al., 1990). Cyr61 has recently been described to be downregulated in approximately half of the cases of prostate cancer (Pilarsky et al., 1998). In order to investigate the role of steroid-induced trans- criptional events in the course of Cyr61 expression, we performed serum starvation on three human prostate car- cinoma cell lines which was followed by the addition of different steroids to the culture medium. The mRNA of the cells was analysed for Cyr61 expression by a comparative multiplex RT-PCR. MATERIAL AND METHODS The human prostate cancer cell lines PC-3, DU145, and LNCaP were cultivated according to the supplier’s recommendation (ATCC). Phenol red was omitted from the medium for at least 5 passages to avoid any ex- ternally induced steroid receptor expression. Serum starvation was performed for 24h with 0.5% fetal bovine serum (FBS) instead of 10% FBS. The cells were then fed with the appropriate medium containing 0.5% FBS and 20nM steroid. The steroids investigated here were: dexa- methasone (Dex), progesterone (Prog), estradiol (E2), and dihydrotestosterone (DHT). The experiment was stopped after 30min, 60min, and 480min by decanting the culture medium and lysing the cells in a buffer containing GTC. The mRNA was prepared by use of the QuickPrep Micro- Kit (Pharmacia, Uppsala). All experiments were perfomed in triplicate. DNA contamination was removed by DNase I digestion. The comparative multiplex RT-PCR was performed as previously described (Pilarsky et al., 1998). Briefly, RT is performed by using the Ready-to-go beads in combi- nation with oligo-d(T)-primers. For the PCR, there were used primers for glyceraldehyde 3-phosphate dehydro- genase (GAPDH) and for the 3’-UTR of the transcript of CYR61 as previously described (Pilarsky et al., 1998). All PCRs were analysed by electrophoresis on 6% dena- turing polyacrylamide gels. CONCLUSIONS RESULTS Cyr61 exhibits different patterns of expression in all three tested cell lines upon stimulation with steroids after serum starvation. Cyr61 has been described to be an immediate early gene in mouse fibroblasts. This does not seem to apply to the prostate cancer cell lines LNCaP and DU145. Therefore, we suggest a different role for Cyr61 in epithelial cells. The presence or absence of the androgen receptor seems not to be sufficient to explain the ob- served expression pattern in the tested cell lines. It is likely that the action of the steroids is mediated by the glucocorticoid receptor as there can be observed an expression after stimulation with four different kinds of steroids. The altered Cyr61’ might be of potential interest to determine the recently described metastasis promoting effects of Cyr61 (Kolesnikova and Lau, 1998). However, it will be interesting to correlate the findings obtained in the described experiments to the metastatic potential of tumor material of patients. Serum starvation for 24h did not result in the expression of the wild-type Cyr61 in LNCaP cells. There can be detected a slightly shorter transcript (Cyr61’) at time point t=0. Addition of serum diminishes the expression of this transcript whereas the addition of the pure steroids augments the expression of Cyr61’. All steroids induced a similar transcriptional response for Cyr61. After 24h of serum starvation, there is no transcript of Cyr61 detectable. The highest level of expression after 30min can be induced by estradiol followed by dihydrotestosterone. All steroids exhibit an effect after 60min and to a minor extent after 480min. For some conditions an additional band (Cyr61’) below the original Cyr61 band has been observed. LNCaP cells PC-3 cells There was no expression observed in DU145 cells after 24h of serum starvation and after 30min of stimulation with steroids. After 60min of stimulation, the most remarkable effect is achieved by progesterone. The effect is still prominent after 480min of stimulation. Note that the minor band intensity of Cyr61 after 480min is due to a smaller amount of charged PCR sample as can be judged from the glyceraldehyde 3-phosphate dehydrogenase band. DU145 cells 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 p GAPDH Cyr61 GAPDH Cyr61 p 1 2 3 4 5 6 7 8 9 10 11 12 13 Cyr61 GAPDH 0:t=0 1-3:serum for 30, 60, 480min 4, 8, 12: E2 for 30, 60, 480min 5, 9, 13: DHT for 30, 60, 480min 6, 10, 14: Prog for 30, 60, 480min 7, 11, 15:Dex for 30, 60, 480min p: Cyr61 plasmid 1:t=0 2, 6, 10:E2 for 30, 60, 480min 3, 7, 11: DHT for 30, 60, 480min 4, 8, 12: Prog for 30, 60, 480min 5, 9, 13:Dex for 30, 60, 480min p 1 2 3 4 5 6 7 8 9 10 11 12 13 p: Cyr61 plasmid 1:t=0 2, 6, 10:E2 for 30, 60, 480min 3, 7, 11: DHT for 30, 60, 480min 4, 8, 12: Prog for 30, 60, 480min 5, 9, 13:Dex for 30, 60, 480min