Effect of selenite on cell surface fibronectin receptor

@)Copyright 1994 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/94/4601-2-0079 $06.20

Effect of Selenite on Cell Surface Fibronectin Receptor

LIN YAN AND GERALD D. FRENKEL*

Department of Biological Sciences, Rutgers University, Newark, NJ 07102

Received November 23, 1993; Accepted January 20, 1994

ABSTRACT

Incubation of cells with selenite, under conditions in which there is no effect on cell viability, results in a decrease in the rate of their subsequent attachment to extracellular matrix proteins such as fibronectin (1). The attachment was inhibited by a pentapeptide containing the RGD sequence and by antibody against the cellular fibronectin receptor (0~5~1 integrin), indicating that it is receptor-mediated. To investigate whether exposure to selenite has an effect on fibronectin receptors, we assayed for their presence on the cell surface by measuring the ability of cells to attach to a surface that had been coated with antibodies to the receptor. Brief exposure of cells to low concentrations of selenite resulted in a significant decrease in their ability to attach to monoclonal antibodies against the ~5 or 61 subunits of the fibronectin receptor, as well as to potyclonal antibodies against the complete receptor. This indicates that exposure to selenite results in a decrease in receptors that are present at the cell surface. Exposure of the cells to selenate, selenocystine or selenomethionine did not result in a significant decrease in cell surface receptors. Pre- incubation of the cells with selenite was required for the effect, indicating that selenite does not directly interfere with receptor structure or function.

Index Entries: Selenium; fibronectin receptor; integrin; HeLa cells.

INTRODUCTION

Tumor cells possess surface receptors for Extracellular Matrix (ECM) proteins such as f ibronectin (2). These receptor proteins, k n o w n as inte-

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 79 Vol. 46, 1994

80 Yan and Frenkel

grins, have been shown to mediate cell attachment to the ECM and base- ment membranes (3,4). Several lines of evidence have indicated that they play a critical role in the processes of invasion and metastasis that are hallmarks of the malignant phenotype (5).

Selenium compounds have been shown to inhibit tumor formation in animals that have been exposed to chemical carcinogens (6). Several hypotheses have been proposed for the mechanism of this anticarcino- genic effect, but no definitive conclusion has been reached (7). In the course of our investigation of this problem, we discovered that exposure of cells to selenite results in a decrease in the rate of their attachment to ECM proteins such as fibronectin (1). This effect of selenite occurred at concentrations and exposure times that caused no other detectable cyto- toxic effects (1). Our initial results led us to hypothesize that the decreased rate of attachment of selenite-exposed cells results from an effect of the compound on the cell surface receptors. We have investigated this hypothesis by assaying for the presence of these receptor molecules at the surface of control and selenite-exposed cells. The results of this investigation, which are reported in this paper, indicate that exposure of cells to selenite results in a decrease in the fibronectin receptor molecules that are present at the cell surface.

MATERIALS AND METHODS

Materials

Bacteriological multiwell (96-well) plates were obtained from Corn- ing (Coming, NY). Sodium selenite was purchased from Gallard- Schlesinger (Carle Place, NY). Fetal calf serum and Dulbecco's Modified Eagle's Medium were from Gibco (Grand Island, NY). Sodium selenate, seleno-DL-methionine, seleno-DL-cystine, and human fibronectin were obtained from Sigma (St. Louis, MO). Peptides, polyclonal rabbit antibody against fibronectin receptor (0~5~1 integrin), and monoclonal antibody against ~5 integrin subunit were obtained from Telios (San Diego, CA). Monoclonal antibody against [31 integrin subunit, control monoclonal antibody (against N. gonorrhea) and nonimmune rabbit IgG were obtained from Chemicon (Temecula, CA).

Cel ls

HeLa cells were obtained from the American Type Culture Collec- tion (Rockville, MD). They were grown in Dulbecco's Modified Eagle's Medium with 10% heat-inactivated fetal calf serum at 37~ in a 5% CO2 atmosphere.

Cell A t tachment

Multiwell plates were coated with 100 ~L of a solution of fibronectin (0.25 ~g/well) as described previously (1). Coating with antibodies was

Biological Trace Element Research Vol. 46, 1994

Selenite and Fibronectin Receptors 81

carried out by incubation of the well overnight at 4~ with 100 ~tL of a solution of the antibody in 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1. In all experiments 0.2 mL of a cell suspension, containing 1 x 105 cells, was added to a microwell, and after incubation at 37~ the number of attached cells was determined as follows: The wells were rinsed twice with 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1 and the cells that remained attached to the well were removed with trypsin and counted using a hemocytometer.

RESULTS

We previously reported that incubation of cells with selenite resulted in a decrease in the rate of their attachment to fibronectin, and we hypothesized that this could result from an effect of selenite on cell surface fibronectin receptors (1). To examine this hypothesis we first determined whether the attachment of these cells to fibronectin does in fact involve the receptor. Cells were incubated with (polyclonal) antibody against fibronectin receptor (~5 ~1 integrin) and their ability to attach to fibronectin was examined. The results (Fig. 1) show that the antibody inhibited attachment, whereas nonimmune rabbit IgG did not. Similarly, incubation of the cells with a peptide containing the RGD sequence, which has been shown to specifically inhibit receptor-mediated attachment (8), resulted in a decrease in their attachment to fibronectin, whereas incubation with a control peptide that lacks the critical sequence had no effect on attachment (Fig. 1).

These results indicate that the attachment of these cells to fibronectin is mediated by the fibronectin receptor. We hypothesized that exposure of the cells to selenite causes a decrease in cell-surface receptors that results in a decrease in their rate of attachment to fibronectin. To examine this hypothesis, we measured cell surface receptors with a modifica- tion of the "panning" technique that has been utilized extensively in the fractionation of cells based on their surface antigens (9). This assay measures the presence of proteins at the cell surface by determining the ability of cells to attach to a solid surface that has been coated with antibodies to the protein. As shown in Fig. 2 and 3, cells were able to attach to wells that had been coated with monoclonal antibodies against the 0~5 and ~1 subunits of the receptor, but not to wells coated with an isotype-matched monoclonal antibody raised against an unrelated protein. Furthermore, attachment to the receptor antibodies was inhibited by preincubation of the cells with the homologous antibody but not the unrelated antibody.

This assay for cell surface proteins was used to examine whether exposure of cells to selenite alters the number of fibronectin receptors at the cell surface. Cells were exposed to various concentrations of selenite for 2 h and the ability of cells to attach to the antibody-coated wells was examined. As shown in Fig. 4, there was a dose-dependent decrease in


82 Yan and Frenkel

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Fig. 1. Attachment of cells to fibronectin. Wells were coated with either fibronectin or bovine serum albumin (0.25 ~tg/well) overnight at 4~ A suspension of 1 x 105 cells was added to the well; after incubation for 3 h at 37~ the number of attached cells was determined as described in Materials and Methods. The results are presented as the percentage of the seeded cells that were attached. (Selenite): Cells were incubated with 15 ~ selenite for 2 h prior to trypsinization and seeding for attachment; (GRGDS), (GRADSP): cells in suspension were incubated with the indicated peptide (5 mg/mL) for 10 rain prior to seeding for attachment; (Ab): cells in suspension were incubated with (polyclonal) antibody to fibronectin receptor (50 ~tL/mL) for 10 min prior to seeding for attachment; (IgG): cells in suspension were incubated with nonimmune rabbit IgG (50 ~tL/mL) for 10 min prior to seeding for attachment.

cell a t tachment to both antibodies at concentrat ions comparable to those that inhibi ted their a t tachment to fibronectin (see Fig. 1 and ref. 1). This result indicates that exposure to selenite causes a decrease in the presence of both receptor subuni ts at the cell surface.

A n alternative explanat ion for the above result is that selenite induces a small change in the specific epi topes of the receptor subuni ts against which the monoclonal antibodies are directed, and that this change results in failure to b ind to the antibodies. This possibility can be


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Fig. 2. Attachment of cells to monoclonal antibodies against fibronectin receptor subunits. Wells were coated overnight at 4~ with 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1 containing various amounts of monoclonal antibody against either ~5 (�9 or 61 (0) integrin subunits. A suspension of I x 105 cells was added to the well, and the number of attached cells was measured after incubation for 1 h at 37~

ruled out by examining cell attachment to polyclonal antibodies against the fibronectin receptor, since the lack of binding to a polyclonal antibody cannot be the result of a small change in the protein antigen. As shown in Figs. 5 and 6, cells attached to wells coated with polyclonal antibody against the ~5 ~1 integrin, but not to non immune IgG, and this attachment was inhibited by homologous antibody but not by control IgG. Thus, with polyclonal antibody as well, this assay measures the presence of fibronectin receptor at the cell surface. Preincubation of the cells with selenite resulted in a dose-dependent decrease in their attachment to the polyclonal antibody (Fig. 7). This result clearly demonstrates that exposure to selenite results in a decrease in fibronectin receptor molecules that are present at the cell surface.

We previously showed that several other selenium compounds, including selenate, selenomethionine, and selenocystine, do not inhibit cell attachment to fibronectin (1). We have now found that none of these compounds have any effect on cell surface fibronectin receptors (Fig. 7). This is consistant with the hypothesis that the inhibitory effect of selenite on cell attachment to fibronectin is a result of its effect on cell surface receptors.


84 Yan and Frenkel

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Fig. 3. Attachment of cells to monoclonal antibodies. Wells were coated with monoclonal antibody against either ~5 or ~1 integrin subunits or with a control monoclonal antibody (0.25 ~tL of antibody in 100 ~tL of 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1). A suspension of 1 x 105 cells was added to the well, and the number of attached cells was measured after incubation for 1 h at 37~ The results are presented as the percentage of the seeded cells that were attached. (mAb): Cells in suspension were incubated with the indicated monoclonal antibody (25 ~tL/mL) for 10 min at 37~ prior to seeding for attachment; (IgG): cells were incubated with control monoclonal antibody (25 ~tL/mL) for 10 rain prior to seeding.

A decrease in cell surface receptors could be the result of a direct effect of selenite on the proteins, or an effect on their synthesis or deposition at the cell surface. If the first possibility is correct then we w o u l d expect that selenite could prevent cell a t tachment to antibodies w h e n it is present dur ing the a t tachment process. We have found, however , that pre incubat ion of the cells wi th selenite was required for the decreased presence of receptors at the cell surface (Table 1). This result indicates that selenite is most likely not affecting existing receptors, bu t rather the deposi t ion of new molecules at the cell surface (see Discussion).


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Fig. 4. Effect of preexposure to selenite on cell surface fibronectin receptor subunits. Cells were incubated for 2 h with the indicated concentrations of selenite, trypsinized and seeded in wells coated with monoclonal antibody against 0~5 (�9 or ~1 (@) integrin subunits (0.25 ~tL of antibody in 100 ~tL of 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1). The number of attached cells was determined after incubation for 2 h. The results are presented as the percentage of the seeded cells that were attached.

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Fig. 5. Attachment of cells to polyclonal antibody, against fibronectin receptor (0~5~1 integrin). Wells were coated by incubation with 100 ~tL of 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1 containing various amounts of antibody against fibronectin receptor. A suspension of 1 x 105 cells was added to the well; after incubation for 2 h at 37~ the number of attached cells was determined.

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86 Yan and Frenkel

Fig. 6. Attachment of cells to antibody and control IgG. Wells were coated either with polyclonal antibody against fibronectin receptor or with nonimmune rabbit IgG (0.25 ~tL of antibody in 100 ~tL of 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1). A suspension of 1 x 105 cells was added to the well, and the number of attached cells was determined after incubation for 2 h at 37~ The results are presented as the percentage of the seeded cells which were attached. (Ab): cells in suspension were incubated with antibody (50 ~tL/mL) for 10 min prior to seeding for attachment; (IgG): cells in suspension were incubated with nonimmune rabbit IgG (50 ~tL/mL) for 10 min prior to seeding for attachment.

DISCUSSION

The attachment of malignant cells to extracellular matrix proteins via cell surface receptors (integrins) is the initial step in the processes of cell invasion of surrounding tissue and metastasis (10). There have been a number of studies that have demonstrated a correlation between the inhibition of integrin-mediated attachment and the inhibition of invasion a n d / o r metastasis. For example, peptides containing the RGD amino acid sequence, which inhibit cell attachment to fibronectin in vitro, also inhibit cell invasion in vitro and experimental metastasis in vivo (11-13). Similarly, dissemination of tumor cells in mouse tissue after iv injection

Biological Trace Element Research Vol, 46, 1994


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Fig. 7. Effect of preexposure to selenium compounds on cell surface fibronectin receptors. Cells were incubated for 2 h with the indicated concentrations of either selenite (�9 selenate (O), selenomethionine (D), or selenocystine (ll). The cells were then trypsinized and seeded in wells coated with polyclonal antibody to fibronectin receptor (0.25 pL of antibody in 100 ~tL of 50 mM sodium phosphate buffer (pH 7.6) containing 150 mM NaC1); the number of attached cells was determined after incubation for 1 h at 37~ The results are presented as the percentage of the seeded cells that were attached.

has been shown to be inhibited by simultaneous injection of an RGD- containing peptide (11,14,15). In addition, monoclonal antibodies against integrin subunits inhibit both cell at tachment and experimental metastasis (16,17). For this reason, our finding that exposure to selenite results in a decrease in cell surface fibronectin receptor suggests that it may also result in a decrease in cell invasiveness. We have obtained evidence that this is in fact the case (18).

The level of receptor molecules at the cell surface is a function of a number of cellular processes, including synthesis, movement to the surface, endocytosis, and degradation (19). An effect of selenite on any of these processes could theoretically result in a decrease in the number of cell surface receptors. However, the rapid kinetics of selenite action (2 h or less) suggests that the most likely mechanism is an inhibitory effect on the deposition of integrin proteins at the cell surface. An active endocy- totic process results in a half life of cell-surface integrin molecules of less than 20 min (20,21); thus inhibition by selenite of the placement of new integrins at the surface could result in a rapid decrease in the number of cell surface molecules. We are currently investigating whether exposure of cells to selenite results in a change in any of the processes that are


88 Yan a n d Frenkel

Table 1 Effect of Selenite on Cell Surface Fibronectin Receptora

Cells attached x 10 -4

Matrix No Se Se preexposure Se during attachment

pAb [~5~1] 7.1 _+ 1.3 1.8 _+ 1.1 5.6 _+ 0.5 mAb [c~5] 5.9 _+ 0.6 2.6 _+ 0.8 5.4 _+ 0.4 mAb [~1] 6.2 _+ 0.05 1.7 _+ 0.1 6.2 _+ 0.7

aCells were either preexposed to 15 ~M selenite for 2 h prior to testing for attachment to the antibodies, or the selenite was present during the attachment. pAb: polyclonal antibody; mAb: monoclonal antibody.

k n o w n to be requ i red for the depos i t i on of in tegr in molecu les at the cell surface.

ACKNOWLEDGMENTS

We t h a n k F. Abdul laev, E. Bonder, P. Caffrey, D. Ganea, Y. Gong , and C. MacVicar for he lpfu l discussions . This w o r k was s u p p o r t e d by Gran t ES-04087 f rom the Nat iona l Inst i tu tes of Heal th . This is pub l i ca t ion n u m - ber 122 f rom the D e p a r t m e n t of Biological Sciences, Ru tge r s -Newark .

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Selen i te a n d Fibronect in Receptors 89

14. R. J. Tressler, R N. Belloni, and G. L. Nicolson, Cancer Commun. 1, 55-63 (1989).

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17. H. P. Vollmers, I. S. Braun, C. A. Waller, V. Schirrmacher, and W. Birchmeier, FEBS Lett. 172, 17-20 (1984).

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Effect of selenite on cell surface fibronectin receptor

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