SHORT NOTE
Specific Binding of [3H]Heparin to Human Carcinoma SW-13 and Other Mammalian Cells
JAROSLAVA HALPER
Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
This study reports on the specific binding of [3H]hepa- 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 90 and 120 min at 22℃. Scatchard analysis revealed two classes of binding sites. The apparent Ka for high-affinity receptors is 2.14 × 10-8 M with 1.48 X 106 sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin. @ 1990 Academic Press, Inc.
INTRODUCTION
Heparin, a potent anticoagulant, acts as a dual modu- lator of growth [1, 2]. 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 [4-8]. Several laboratories have demonstrated specific binding of heparin to mam- malian cells both nontransformed [9-13] 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 [1]. 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.
MATERIALS AND METHODS
Materials. High-molecular-weight (HMW) heparin (MW 12,000) from porcine intestinal mucosa, low-molecular-weight (LMW) hepa- rin (MW 6,000), neutral dextrans (MW 40,200 and 9,400), dextran sulfate (MW 8,000), hyaluronic acid, and insulin were purchased from Sigma Chemical Co. (St. Louis, MO). HMW heparin derived from por- cine intestine (MW 14,000), bovine lung (MW 12,000), and LMW hep- arin (MW 3,000) were from Calbiochem Brand Biochemicals (San Diego, CA). Radiolabeled [3H]heparin (specific activity 0.30 mg/mCi;
MW 6,000-20,000) was obtained from New England Nuclear (Bos- 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 5a with 5% calf serum (CS) at 37℃ in a humidi- fied atmosphere of 5% CO2 and 95% air. Mouse embryo AKR-2B cells [16], human fibrosarcoma HT1080 [17], and human rhabdomyosar- coma A204 [18] were cultured in McCoy’s medium 5a supplemented with 5% fetal bovine serum (FBS). Mouse fibroblasts Balb/c3T3 [19], bovine kidney MDBK [20], and mink lung Mv1Lu CCL64 cells [21] 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].
[3H]Heparin binding assay. Cells (2 X 105 or 105 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 cells were first washed three times with phosphate- buffered saline (PBS). Then 1 or 0.75 ml binding buffer consisting of 0.1% bovine serum albumin and 5 mM MgCl2 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 × 10-6 or 10-5 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 ng/ml (approximately 25,000 to 36,000 cpm/ml). The plates were incubated at 22℃ for 90 to 120 min on a rocker platform. The time course of heparin binding was determined at several time points. The effect of temperature was examined at 22, 37, and 4℃. 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 ul of lysis 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 a 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- mocytometer. Scatchard analysis [24] was performed with the ligand- binding curve-fitting program [25] according to Munson and Rod- bard [26].
RESULTS
Equilibrium of [3H] Heparin Binding to SW-13 Cells
Scatchard analysis revealed two classes of binding sites. An apparent Ka between 8.96 × 10-9 and 1.4 × 10-8 M (average value 2.14 × 10-8 M) with binding sites be-
B/F
0.047
Kd 1.4 x10-8 M
0.023
Kd 3.1x10 M
0.000
0
1.64
3.28
4.93
Bound (nM)
tween 8.6 × 105 and 2 X 106 sites per cell (Fig. 1, Table 1) for high-affinity sites was calculated by using the ligand- binding curve-fitting program [25] by Munson and Rodbard [26]. At the point of maximal binding between 90 and 120 min (see below), on the average 3.5% of the added [3H]heparin was specifically bound to 3.9 X 105 cells present on the average in a well. Nonspecific bind- ing was 5% or less of total binding. Preliminary data show that low-affinity sites had an apparent Ka between 2.7 × 10-6 and 2.28 × 10-5 M.
Specificity of [3H ] Heparin Binding to SW-13 Cells
The binding of heparin to SW-13 cells is saturable. Increasing concentrations of unlabeled high-molecular
140
Percent 3H Heparin Bound
120
100
80
60
HMW Heparin
40
LMW Heparin
Dextran Sulphate
20
Neutral Dextran
Insulin
0
0
10
10
10
9
10-8
10
7
10 6
10
5
Mol Competitor
heparin led to increasing displacement of radiolabeled heparin (Fig. 2). Three different preparations of HMW heparins, two different LMW heparins, two neutral dex- trans, dextran sulfate, hyaluronic acid, and insulin were tested for their ability to inhibit [3H]heparin binding to SW-13 cells. All three HMW heparins and dextran sul- fate were equal in their ability to displace radiolabeled heparin. Fifty percent displacement occurred between 10-8 and 3 × 10 8 M unlabeled heparin or dextran sul- fate, whereas specific binding of [3H]heparin was inhib- ited 99-100% at 10-6 M unlabeled heparin or dextran
| Cell line | Origin | Ka (M)ª | Bmax (M)b | Binding sites per cell |
|---|---|---|---|---|
| SW-13 | Human carcinoma of adrenal cortex | 2.14× 10-8 | 5.72 × 10-10 | 1.48 × 106 |
| ± 1.4 ×10-8 | ± 2.96 × 10-10 | ± 0.5 × 106 | ||
| A204 | Human rhabdomyosarcoma | 5.16 × 10-9 | 1.91 × 10-10 | 7.1×105 |
| ± 2.4 ×10-9 | ± 1.46 × 10-10 | ± 5.8 X 105 | ||
| HT1080 | Human fibrosarcoma | 2.65 × 10-9 | 9.2 × 10-11 | 1.48 × 105 |
| ± 0.92 × 10-9 | ± 2.5 × 10-11 | ± 0.3 ×105 | ||
| MDBK | Bovine kidney | 6.88 × 10 -9 | 8.4 × 10-11 | 1.68 × 105 |
| ± 0.3×10-10 | ± 1.4 × 10-11 | ± 0.3 × 105 | ||
| Mv1Lu | Mink lung | 7.72 × 10-9 | 2.08 × 10-10 | 4.17 X 105 |
| ± 2.9 × 10-9 | ± 1.2 × 10-10 | ± 2.5 ×105 | ||
| Balb/c3T3 | Mouse fibroblasts | 2.7×10-8 | 4.56 × 10-10 | 1.4 ×106 |
| ± 0.6×10-8 | ± 1.8 ×10-10 | ± 0.9 × 106 | ||
| AKR2B | Mouse fibroblasts | 7.83 × 10-9 | 4.14 × 10-10 | 6.9 ×105 |
| ± 0.8 ×10-9 | ± 2.2 ×10-10 | ± 4.1X105 |
Note. The data for each cell line represent a mean of three assays (+1 standard deviation) performed for 90 min at 22℃ as described under Materials and Methods.
” Dissociation constant.
৳ Binding capacity.
sulfate (Fig. 2). Hyaluronic acid (data not shown), both neutral dextrans, and insulin displayed no significant binding even at 10-5 M concentrations. Both LMW hep- arins showed intermediate binding (Fig. 2).
Time- and Temperature-Dependent [3H] Heparin Binding
Specific heparin binding was studied at 22, 37, and 4℃ at intervals specified below. At room temperature, 14 to 30% of maximal specific binding occurred within the first minute of binding. Maximum binding was achieved between 90 and 120 min of incubation time lasting up to 5 h in most assays. However, at incubation times beyond 2 h we usually observed progressive rounding up and de- tachment of cells. At 37℃ the time course was similar with 20 to 33% maximum specific binding occurring at 5 min and maximum between 1 and 2 h. Again cells started rounding up and detaching at incubation times over 2 h. At 4℃, maximum binding occurred at 2 h, although only at 60% of maximum binding at 37℃ in replicate plates assayed at the same time (data not shown).
[3H ] Heparin Binding to Other Cell Lines
We tested the binding of [3H]heparin to several cell lines, mouse embryo AKR-2B, mouse Balb/c3T3 fibro- blasts, human fibrosarcoma HT-1080, human rhabdo- myosarcoma A204, bovine kidney MDBK, and mink lung Mv1Lu (CC164) cell lines. Binding occurred to all of these cell lines. Table 1 shows apparent Ka values for high-affinity sites in these cells. Calculations revealed a second class of low-affinity sites for all examined cell lines, with the exception of Balb/c3T3 cells which pos- sess only one class of high-affinity receptors (data not shown).
DISCUSSION
We have shown that [3H]heparin binds specifically to human adrenocortical carcinoma SW-13 cells and sev- eral other mammalian cell lines. Scatchard analysis re- solved heparin binding into two receptor classes for all cell lines examined except for Balb/c3T3 cells which possess only one class of receptors. The apparent Ka for high-affinity binding sites was between 2.6 X 2.69 × 10-9 M (for HT1080 cells) and 2.7 X 10-8 M (for Balb/c3T3 cells). The number of high-affinity receptor sites ranged from 1.4 X 105 sites per HT1080 cell to 1.48 X 106 sites per SW-13 cell. These results are similar to data from other studies on heparin binding [9-14]. Monocytes, platelets, and hepatocytes bind heparin with an appar- ent Ka of about 10-7 M and possess about 106 binding sites per cell [9, 12, 13]. Chinese hamster ovary cells bind heparin with a Ka of 5 X 10 7 M with 2 X 106 binding
sites per cell [10]. Vascular smooth muscle cells possess 105 binding sites per cell with a Ka of 10-9 M [11]. B-16 melanoma cells bind heparin with a Ka of 2-5 X 10-8 M with 0.5 X 107 binding sites per cell [14]. In contrast to our studies, these investigators have reported only one class of receptors. However, Horne and Chao report that Scatchard analysis resolved heparin binding into two classes of receptors on activated, but not resting, plate- lets [27]. Castellot et al. [11] attributed the appearance of a second, low-affinity class of receptors to impurities present in commercial preparations of radiolabeled hep- arin. They reported one class of receptors when using heparin radiolabeled in their laboratory. We cannot ex- clude the presence of impurities in commercially radiola- beled heparin used in this study as being responsible for the appearance of low-affinity receptors. However, Balb/c3T3 cells exhibited only one class of receptors in this study, and similarly, heparin binding to monocytes, platelets, and B-16 melanoma cells occurred to a single class of receptors when commercial preparations of ra- diolabeled heparin were used in other studies [10, 12- 14]. So the possibility cannot be excluded that different cells, cell types, or the same cell in a different stage of activation [27] possess one or several classes of recep- tors. Heparin is known to be a heterogenous mixture of negatively charged glycosaminoglycans of variable mo- lecular weight. It is, therefore, possible that two or more different subspecies or fragments of heparin bind to membrane receptors with different affinity and that binding sites are not very specific for heparin since an- ionic dextran sulfate, a compound with a different sac- charidic backbone, competes for binding of heparin as a function of its charge.
In agreement with Sobel and Adelman [12], HMW heparin and dextran sulfate bind to cells (be they SW- 13 cells or platelets) with equally high affinity, LMW heparin binds with intermediate affinity, and neutral dextrans and hyaluronic acid do not bind at all.
Results from our laboratory and others indicate that heparin binds to specific cell membrane receptors on a variety of cell types, including human tumor cells. This is in good correlation with data showing that heparin acts as a growth modulator for a variety of cells, both as a growth inhibitor [1, 4-8] and as a growth stimulator [2]. Future studies are necessary to characterize inter- nalization and intracellular processing of heparin and to elucidate the relationship between the heparin binding and heparin modulation of growth of SW-13 cells. More studies are also required to elucidate the problem of low- affinity binding sites on some cell lines.
I thank Dr. Duncan Ferguson for useful discussions and Bobbie Car- ter for technical help. This project was supported by NIH Grant CA44039 and by a grant from the Veterinary Medical Experiment Sta- tion, University of Georgia.
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Received September 19, 1989
Revised version received December 21, 1989
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