Pakistan Journal of Pharmaceutical Sciences Vol. 18, No.3, July 2005, pp.46-48 INHIBITION OF PROTEINASE 3 (PR3) BY SURAMIN AND FETAL CALF SERUM (FCS): EFFECT OF PR3 AND SURAMIN ON CHINESE HAMSTER OVARY CELLS (CHO-CELLS) GHOLAMREZA ASADI KARAM, MOHAMMAD JAVAD RASAEE*, MEHDI MAHMOODI AND MOHAMMAD KHAKSARI** Department. of Biochemistry, Faculty of Medicine, Rafsanjan Medical University, Post code: 7719617996, Rafsanjan, Iran *Department. of Biotechnology, Faculty of Medicine, Tarbiat Modarres University Tehran, Iran **Department of Physiology, Faculty of Medicine, Kerman Medical University, Kerman, Iran Background: Proteinase 3 (PR3) is a lysosomal protease that is stored in azurophilic granules neutrophilic granulocytes and monocytes. A number of inhibitors for this proteinase are reported. Comprehensive studies on the inhibitory effect of suramin and heat treated fetal calf serum (∆FCS) on PR3 have not been reported. It has been reported that PR3 is able to destroy the cytoskeletal integral proteins, but we have not find any reports which showed the effect of this protease on Chinese hamster ovary cells (CHO-cells) in culture medium. Suramin has proven to be useful as an antitumor drug, but there was not any report on the effect of suramin on CHO-cells. Methods: The effects of various concentrations of ∆FCS (from 0.5% up to 10%) and suramin (from 0.8 µM up to 100 µM) on PR3 and different concentrations of suramin (from 0.8 µM up to 1000 µM) on CHO-cells were investigated. Data analysis were performed by, Kolmogorov-Smirnov test, ANOVA test and Tukey HSD post tests. Results: Results showed that ∆FCS and suramin have an inhibitory effect on PR3 and these effects increased with increasing the concentration significantly (p < 0.01). PR3 with the concentration of 2.2 Unit/ml has no effect on CHO-cells. Although suramin with the concentration of less than 125 µM cell growth retarded for only a few hours, but with the concentration of 125 to 250 µM inhibit the cell growth for a week, and after that cells gain normal growth gradually. Suramin with concentration of more than 500 µM inhibited the cell growth completely. Conclusions: Although suramin reversibly inhibit the PR3 activity but in concentration of less than 250 µM it had no long-term effect on CHO-cells. Therefore it can be used in the investigation of proteases. There were unknown components in ∆FCS, which cause the inhibition of PR3 activity. This finding is very important in PR3 production in culture medium. However CHO-cells are resistant to PR3 and suramin in low concentration. Keywords: Proteinase 3, suramin, FCS, CHO-cells. INTRODUCTION Proteinase 3 (PR3) is a human neutrophil serine protease that is a target of classical antineutrophil cytoplasmic autoantibodies (c-ANCA) in sera from patients suffering from autoimmune systemic vasculitis conditions such as Wegener’s Granulomatosis (Rao et al. 1996; Rao et al.1991). This enzyme is a lyzosomal proteinase stored in azurophilic granules, neutrophilic granulocytes and monocytes (Rao et al.1991 ). This enzyme is able to destroy the cytoscletal integral proteins (Rao et al. 1996; Leid et al.1993). The natural inhibitor of PR3 in human body is found to be α-1-antitrypsin (Jenne,1994). In addition a number of synthetic inhibitors for this proteinase have been reported (Kam et al.1992 ). The inhibitory effect of suramin as an antitumor drug has been described by prevention the growth factors binding to the cancerous cell surface (Voogd et al., 1993). Cadene et al (1997) explained reversible inhibitory effect of suramin on PR3 activity. Although Ballieux et al. (1994) investigated the inhibitory effect of heat treated fetal calf serum (∆FCS) on PR3 binding to human umblical vein endothelical cells (HUVEC) , but there was no report on the effect of ∆FCS on PR3 which may be important on the culture condition specially when PR3 is being cloned and expressed in eukaryotic cells such as Chinese Hamster Ovary cells (CHO-cells). The aim of this study was to evaluate the following: 1) Can ∆FCS inhibit the proteinase activity of PR3 ? 2) How is inhibitory effect of different concentration of suramin on PR3 activity ? 3) What is the effect of PR3 as a proteinase on growth and proliferation of CHO-cells ? 4) In what manner CHO-cells behavior is in presence of different concentration of suramin ? In order to find possible answers to the above questions, we determined the inhibitory effect of different concentration of suramin and ∆FCS on PR3 activity and different concentration of suramin on the growth and Correspondent: Dr. Gholamreza Asadi Karam, Dept. of Biochemistry, Faculty of Medicine, Post code: 7719617996 Rafsanjan, Iran. Tel: 0391- 522 5400, Fax: 0391-522 5209, E-mail: asadi_ka@yahoo.com