SHORT NOTE Specific Binding of [3H]Heparin to Human Carcinoma SW-1 3 and Other Mammalian Cells JAROSLAVA HALPER This study reports on the specific binding of [‘Hlhepa- rin to human adrenocortical carcinoma cell line SW- 13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between SO and 120 min at 22°C. Scatchard analysis revealed two classes of binding sites. The apparent Kd for high-affinity receptors is 2.14 X lo-* Mwith 1.48 X 1O’sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin. 0 mm *oademio Pr.ss. he. INTRODUCTION Heparin, a potent anticoagulant, acts as a dual modu- lator of growth [l, 21. The anticoagulant activity can be separated from the growth-modulating activity on an affinity antithrombin column [3]. Heparin inhibits the growth of a variety of cells [4-81. Several laboratories have demonstrated specific binding of heparin to mam- malian cells both nontransformed [9-131 and trans- formed [14]. We have shown previously that heparin in- hibits the growth of human carcinoma SW-13 cells in monolayer and soft agar [l]. In the present report we examined the binding of heparin to SW-13 and to sev- eral other cell lines. Heparin binds to all the cell lines examined. The binding of heparin, at least to SW-13 cells, is specific, saturable, and time- and temperature- dependent. MW 6,000-20,000) was obtained from New England Nuclear (Boa- ton, MA). Cell culture. SW-13 cells, derived from a human adrenocortical carcinoma [15], were from Dr. Harold L. Moses and were maintained in McCoy’s medium 5e with 5% calf serum (CS) at 37’C in a humidi- fied atmosphere of 5% CO, and 95% air. Mouse embryo AKR-2B cells [161, human fibrosarcoma HT1080 [17], and human rhabdomyosar- coma A204 [la] were cultured in McCoy’s medium 5a supplemented with 5% fetal bovine serum (FBS). Mouse fibroblasts Balb/c3T3 [19], bovine kidney MDBK [ZO], and mink lung MvlLu CCL64 cells [Zl] were maintained in Dulbecco’s minimal Eagle’s medium with 10% CS. Cells were regularly examined after Hoechst No. 33258 staining to en- sure that they were free of mycoplasms [22]. [3HlHeporin binding assay. Cells (2 X lo5 or lo6 per well) were plated in 6. or 12.well plates, respectively, in 2 or 1.5 ml McCoy’s me- dium 5a with 5% CS. Two to four days later when the cells were 60 to 95% confluent, the cella were first washed three times with phosphate- buffered saline (PBS). Then 1 or 0.16 ml binding buffer consisting of 0.1% bovine serum albumin and 5 m&4 MgCll in PBS [23] was added per well of 6- or 12.well plates, respectively, containing various levels of unlabeled heparins or their competitors in duplicate or triplicate. For nonspecific binding 3 X 10.’ or 10m6 M heparin was added in repli- cate wells. No unlabeled heparin was added to replicate wells for deter- mination of total binding. [3H]Heparin was added to all wells at 82- 87 rig/ml (approximately 25,000 to 36,000 cpm/ml). The plates were incubated at 2YC for 90 to 120 min on a rocker platform. The time course of heparin binding WBB determined at several time points. The effect of temperature was examined at 22,37, and 4’C. The assay was terminated by the removal of the binding buffer and by three washes with PBS. Cells were lysed for at least 20 min with 150 to 250 &I of lyais buffer (0.2 M NaOH, 0.1% sodium dodecyl sulfate, and 0.1% BSA). The cell lysate was added to aqueous scintillation fluid and counted in B scintillation counter. Specific binding was determined by sub- tracting nonspecific binding from total binding. The cell count was determined from replicate wells by counting trypsinized cells in a he- moeytametar. Scatchard analysis [24] was performed with the ligand- binding curve-fitting program [25] according to Munson and Rod- bard (261. MATERIALS AND METHODS RESULTS Materials. High-molecular-weight (HMW) heparin (MW 12,WO) from porcine intestinal mucow., low-molecular-weight (LMW) hepa- rin (MW 6,000). neutral dextrans (MW 40,200 and 9,400). deatrsn sulfate (MW 8,000), hyaluronic acid, and insulin were purchased from Equilibrium of [3H]Heparin Binding to SW-13 Celk Sigma Chemical Co. (St. Louis, MO). HMW heparin derived from par- tine intestine (MW 14,WO).bovine Lung (MW 12,000), andLMW hep- Scatchard analysis revealed two classes of binding arin (MW 3,000) were from Calbiochem Brand Biochemicala (San sites. An apparent Kd between 8.96 X 1O-9 and 1.4 X 10-s Diego, CA). Radiolabeled [3H]heparin (specific activity 0.30 mg/mQ; M (average value 2.14 X lo-’ M) with binding sites be- 324