Modulation of Growth of Human Carcinoma SW-13 Cells by Heparin and Growth Factors
JAROSLAVA HALPER* AND BOBBIE J. CARTER
Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, in- cluding smooth muscle cells, certain fibroblasts, and epithelial cells, and to mod- ulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unre- lated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both mono- layer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin- like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incor- poration in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We pro- pose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.
Heparin acts as a growth inhibitor for several cell types, such as smooth muscle cells (Clowes and Kar- novsky, 1977), mesangial cells (Coffey and Karnovsky, 1985; Castellot et al., 1985b), certain fibroblasts (Paul et al., 1980), rat cervical epithelial cells (Wright et al., 1985; Wright and Karnovsky, 1987), and skin kerati- nocytes (Ristow and Messmer, 1988). The inhibitory activity can be separated from the anticoagulatory ac- tivity by fractionation of heparin on an affinity an- tithrombin-Sepharose column (Guyton et al., 1980). When investigating in previous studies the effect of aFGF on the soft agar growth of SW-13 cells, we ob- served a paradoxical effect of heparin on SW-13 cells. Instead of potentiating mitogenic effects of aFGF, hep- arin inhibited all soft agar growth of SW-13 cells (Halper and Moses, 1987). To explore further this effect of heparin on SW-13 cells, we investigated the inhibi- tory effect of heparin on monolayer and soft agar growth of human carcinoma SW-13 cells (Halper and Carter, 1988). Transforming growth factor type e (TGFe), basic fibroblast growth factor (bFGF), and
acidic FGF (aFGF) are able to overcome this inhibition, whereas epidermal growth factor (EGF) and insulin- like growth factor I (IGF-I) are ineffective. Heparin also modulates the growth of AKR-2B cells, its effect depending on the absence or presence of other growth factors.
MATERIALS AND METHODS Materials
High- and low-molecular-weight heparins were pur- chased from Sigma Chemical Co. (St. Louis, MO) and from Calbiochem Brand Biochemicals (San Diego, CA). EGF was from Bioproducts for Science, Inc. (Indianap- olis, IN) and IGF-I from Imcell Products Division (Terre Haute, IN). All four substances were dissolved in solution A, consisting of 10 mM glucose, 3.0 mM
Received January 5, 1989; accepted May 12, 1989. *To whom reprint requests/correspondence should be addressed.
KCI, 130 mM NaCl, 1.0 mM Na2HPO4.7H2O, 0.0033 mM phenol red, and 30 mM 4-(2-hydroxyethyl)-1-pi- perazineethanesulfonic acid, pH 7.6 (Shipley et al., 1984). aFGF and bFGF from R & D Systems, Inc. (Min- neapolis, MN) were dissolved in H2O with 1 mg/ml bo- vine serum albumin (BSA) as a carrier. Both [3H]thymidine (specific activity 50-90 Ci/mmol) and [35S]methionine (>800 Ci/mmol) were obtained from New England Nuclear (Boston, MA). TGFe was par- tially purified by a sequence of acid-ethanol extraction and conventional and high-performance liquid chroma- tography (Parnell et al., 1989). After evaporation, TGFe was resuspended in 0.004 M HCI containing 1 mg/ml BSA as a carrier. One unit of TGFe was defined as the amount of TGFe necessary to elicit 50% maximal stimulation of soft agar growth of SW-13 cells.
Cell culture
SW-13 cells, derived from a human adrenocortical carcinoma (Leibovitz et al., 1973) and mouse embryo AKR-2B cells (Proper et al., 1982) were obtained from Dr. Harold L. Moses (Vanderbilt University, Nashville, TN). The cells were maintained in McCoy’s medium 5a (Grand Island Biological Co., Grand Island, NY) sup- plemented with 5% (v/v) calf serum (CS) and 5% (v/v) fetal bovine serum (FBS; both from Hazelton Biologics, Inc., Lenexa, KS), respectively, at 37℃ in a humidified atmosphere of 5% CO2 and 95% air. Cells were regu- larly examined after Hoechst 33258 staining to ensure that they were free of mycoplasmas (Chen, 1977).
Soft agar colony stimulation assay
Soft agar assays were performed as a modification of the method described previously (Halper and Moses, 1983). Briefly, 7.5 x 103 SW-13 cells/ml with appro- priate concentrations of heparin and/or growth factors were suspended in 1 ml of 0.4% SeaPlaque agarose (FMC BioProducts, Rockland, ME) in McCoy’s medium 5a with 5% CS and poured on solidified base layers of 1 ml of 0.8% agarose in McCoy’s medium 5a with 5% CS in 35 mm Petri dishes (Falcon). Assays were analyzed at 7-10 days. Colonies with a diameter >50 um were counted using an inverted microscope and an eyepiece equipped with a grid.
Growth curves
On day 0, SW-13 cells were plated at densities 0.5-1 × 104 cells/well in six well (9.6 cm2 well) tissue culture plates (Nunc, Denmark, or Falcon, Becton Dickinson Co., Lincoln Park, NJ) in McCoy’s medium 5a with 5% CS. Twenty-four hours later (day 1), heparin and/or growth factors suspended in small volumes (1%) of an appropriate buffer were added to the cells. On certain days thereafter, cells were trypsinized and counted in a hemocytometer.
[3H]thymidine incorporation
On day 0, SW-13 cells were plated in 24 well culture plates (Nunc or Costar, Cambridge, MA) in McCoy’s medium 5a with 5% CS at a density of 104 cells/well. The next day (day 1), 100 µg/well heparin was added to appropriate wells. Duplicate control wells and wells containing heparin were pulsed with 0.1 pCi/well [3H]thymidine for 24 hr on days 3, 4, 5, 6, and 7. For the last hour of each 24 hr pulse, duplicate wells were
pulsed for 1 hr with 1.0 pCi/well [3H]thymidine on days 4, 5, 6, 7, and 8. The cells were fixed and washed with cold 10% trichloroacetic acid (TCA) and solubilized in 0.2 M NaOH with 0.1% sodium dodecyl sulphate (SDS) and 40 µg/ml herring DNA as a carrier. An aliquot of the solution was added to aqueous scintillation fluid and counted in a scintillation counter.
In a modification of the method described by Shipley et al. (1984), AKR-2B cells that had been plated in 24 well culture plates in McCoy’s medium 5a with 5% FBS at cell density 104 cells/well were arrested as they reached their saturation density by placing them into serum-free (SF) McCoy’s medium 5a for 2 days before the addition of heparin and growth factors. The effect of heparin and growth factors on DNA synthesis was followed at 22-23 hr after the addition of heparin and growth factors by 1 hr pulse with 1.0 pCi [3H]thymidine per well. The cells were fixed and washed with cold 10% TCA and processed as above.
[35S]methionine uptake and protein synthesis
SW-13 cells were plated in 24 well culture plates in McCoy’s medium 5a with 5% CS and heparin added as described for [3H]thymidine. Both [35S]methionine uptake and protein synthesis were evaluated on days 4-7. Cells were washed once with a TBS buffer containing 0.137 M NaCl, 6.7 mM KCI, 0.68 mM CaCl2, 0.5 mM MgCl2, 0.7 mM Na2HPO4, and 25 mM Tris HCI (pH 7.4). Growth factor effect was followed at appropriate times by placing cells into 300-400 ul/well serum- and methionine-free DMEM and puls- ing for 3-4 hr at 37°℃ with 3-10 uCi [35S]methionine per well (Nilsen-Hamilton and Hamilton, 1987). At the end of the pulse, the medium was carefully removed with a Pasteur pipette, so as not to disturb and lyse the cells, and clarified for 5 min in a microcentrifuge. An aliquot of the medium was saved for SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography (see below). The rest of the medium was suspended in 8-10 volumes of cold 10% TCA. The TCA-precipitable material was collected on Metricel filters (Gelman Sciences, Inc., Ann Arbor, MI) according to Shipley et al. (1984). The filters were washed once with cold 10% TCA and twice with cold 95% ethanol, dried, and added to scintillation fluid. Radioactivity was counted in a scintillation counter. The cells were washed twice with TBS buffer immedi- ately after the medium removal, then they were lysed with 100 pl/well cold lysis buffer consisting of 0.5% Triton X-100 (v/v), 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 20 mM Tris HCI, pH 7.5, for 20 min at 4℃ (Scher and Pledger, 1987) and spun for 5 min in a microcentrifuge. An aliquot of the supernatant was added to aqueous scintillation fluid, and radioactivity was counted in a scintillation counter. Another aliquot was saved for SDS-PAGE and autoradiography (see below).
Electrophoresis on SDS-polyacrylamide gels and autoradiography
Aliquots of the clarified medium and/or the superna- tant from the cell lysate pulsed with [35S]methionine were solubilized in an equal volume of reducing Laem- mli sample buffer and boiled for 5 min. The samples were run on discontinuous buffer SDS-polyacrylamide
gels according to Laemmli (1970) using 10-20% acry- lamide gradient. After the run was completed, gels were fixed in 10% acetic acid and immersed in the en- hancing fluorography reagent Amplify (Amersham, Arlington Heights, IL) for 30 min. Then the gels were dried and exposed to X-Omat AR film (Eastman Kodak Co., Rochester, NY) for several hours to several days before being developed.
RESULTS Effects of heparin on soft agar growth of SW-13 cells
Although SW-13 cells form only a few colonies when suspended in soft agar at low densities, they form nu- merous colonies with the presence of either TGFe or FGF in the soft agar culture (Halper and Moses, 1983, 1987). Colony formation to a somewhat lesser degree was noted upon the addition of aFGF from R & D Sys- tems, although no stimulation with aFGF from Bio- medical Technologies, Inc. (Stoughton, MA) was ob- served previously (Halper and Moses, 1987). In the effect of aFGF on soft agar growth of SW-13 cells in our previous studies, we observed a paradoxical effect of heparin on SW-13 cells. Instead of potentiation of mi- togenic effects of aFGF, heparin led to inhibition of soft agar growth of SW-13 cells (Halper and Moses, 1987). When we repeated these experiments later, the inhibi- tion by heparin was even more impressive without the addition of aFGF (Halper and Carter, 1988). Heparin (1-1,000 µg/ml) inhibits the soft agar growth of SW-13 cells almost completely. Because even in the absence of heparin SW-13 cells do not form many colonies it was difficult to determine ED50 for heparin inhibition. However, 50% inhibition occurred at 10µg/ml heparin in soft agar assays with higher colony background (data not shown). Because the soft agar assay precludes the removal of single cells from soft agar, we were un- able to determine whether this inhibitory effect was reversible.
Next we tested whether this inhibitory effect of hep- arin could be overcome by other growth factors known to bind to heparin and known to stimulate soft agar growth of SW-13 cells. We added bFGF, aFGF, and TGFe to heparin (10 µg/ml) and measured the degree of inhibition of colony growth by comparing with colony growth stimulation by bFGF, aFGF, and TGFe only in replicate plates (Table 1). This inhibition persisted to some degree upon the addition of bFGF or aFGF even at doses as high as 10 ng/ml: 10 µg/ml heparin inhib- ited by 60% and 59% soft agar growth stimulation by bFGF and aFGF, respectively. TGFe was also able to counteract the effects of heparin only partially, 63% and 42% inhibition was observed with 3 and 10 U/ml TGFe, respectively, at 10 µg/ml heparin (Table 1). These results also suggest that the action of heparin is cytostatic rather than cytotoxic. To determine whether the effect of TGFe, bFGF, and aFGF was specific or whether other growth factors are able to reverse hep- arin-induced inhibition of soft agar growth of SW-13 cells, we tested EGF and IGF-I in soft agar assays. Neither EGF nor IGF-I had any significant effect on soft agar growth of SW-13 cells, and neither growth factor reversed the growth inhibition by hepa- rin (Table 1).
| Growth factor | No heparin | 10 µg Heparin |
|---|---|---|
| TGFe | ||
| 1 U | 18.6 ± 7.8 | 2.7 ± 2.5 |
| 3 U | 7 ± 4.4 | 2.6 ± 1.7 |
| 10 U | 5.9 ± 2.1 | 3.5 ± 0.95 |
| bFGF | ||
| 1 ng | 3.7 ± 1.4 | 1.7 ± 0.8 |
| 10 ng | 3.8 ± 1.9 | 1.5 ± 0.9 |
| aFGF | ||
| 1 ng | 4.25 ± 0.9 | 0.2 ± 0.2 |
| 10 ng | 4.4 ± 1.35 | 1.8 ± 0.3 |
| EGF | ||
| 1 ng | 1.4 ± 0.8 | 0.24 ± 0.3 |
| 10 ng | 0.85 ± 0.7 | 0.1 ± 0.1 |
| IGF-1 | ||
| 1 ng | 0.7 ± 0.5 | 1.2 ±1.2 |
| 10 ng | 0.5 ± 0.5 | 0.02 ± 0.02 |
1SW-13 cells were seeded at 7.5 x 103 cells/well with heparin and growth factors added in appropriate plates. The results are expressed as the ratio of the number of colonies in plates with a growth factor to the number of colonies in control plates + standard deviation. No colonies were observed in plates with 10 µg/ml heparin only. Colonies larger than 50 um per 20 medium-power (magnification 100 x) fields were counted in quadruplicate plates.
Inhibition of monolayer growth of SW-13 cells by heparin
Because transformed SW-13 cells are very difficult to synchronize and growth arrest in confluent monolayer cultures, we studied the effects of heparin on SW-13 cells in monolayer using sparse cultures of exponen- tially growing SW-13 cells. These studies were per- formed using medium supplemented with 5% CS, be- cause of insufficient growth of cells in preliminary assays with SF medium or medium with decreased se- rum concentration. After plating 0.5-1 x 104 SW-13 cells/well on day 0 and adding heparin with or without growth factors on day 1, we determined the cell number in replicate wells on days 4, 6, 7, or 8 and, in some assays, on days 10 and 12. Heparin (100 µg/ml) inhib- ited the growth of SW-13 cells up to 15% and 14% of the growth of control cells on days 8 and 10, respectively. The inhibition was dose-dependent and persisted for several more days (Fig. 1). The cells cultured with hep- arin appeared otherwise healthy; they remained at- tached to the plastic tissue culture flask and did not undergo any noticeable morphological changes. The heparin-induced inhibition was still apparent on day 10 in some, but not all, assays. To determine whether the inhibition by heparin was reversible, medium from replicate wells of SW-13 cells with or without the con- tent of heparin was discarded on days 2, 3, 4, and 5 after plating. The cells were washed with medium without heparin, and medium without heparin was added for the rest of the experiment. As is shown in Figure 2 the inhibition of growth of SW-13 cells was reversible; however, the process was slow, and it took several days for the cells to recover. The cells remained attached to the bottom of plastic wells and appeared viable at all stages of this experiment when examined microscopically.
Like other investigators before us (Folkman et al., 1983), we have also noted that the inhibitory activity varies among different preparations of heparin. We found that high-molecular-weight heparin from por- cine intestinal mucosa from Calbiochem is more potent
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than high-molecular-weight heparin from bovine lung (Calbiochem) and more potent than high-molec- ular-weight heparin from Sigma. Some lots of low-mo- lecular-weight heparin from Sigma were much more effective than others. The reason for this variability among different lots of heparin is unknown. Increasing cell density abrogated the inhibition of heparin in some assays on day 7 and 10 when faster growing passages of these transformed cells were used in some assays; this has been observed by other investigators as well (Reilly et al., 1986). Similarly, the use of multiwell (12 well) plates with smaller surface area of wells (4.5 cm2/well in 12 well plates vs 9.6 cm2/well in six well plates) led more quickly to confluent monolayer of SW-13 cells and thus to the abolishment of heparin inhibitory ac- tivity as well.
Even in assays with growth inhibition still apparent on day 10, the cell count in wells with heparin was usually higher than the control cell count on day 12. This late “stimulatory” effect probably is indirect, and it might be attributed to at least three factors. First, because of unencumbered growth in control wells, the medium becomes nutrient-depleted more quickly, and thus the cells grow more slowly in the later stages of the assay. Second, cells inhibited by heparin may re- spond by increasing their production and by secreting growth factors able to overcome this inhibition. Third, cells exposed to heparin may be induced to secrete a heparin-degrading enzyme, which eventually would decrease the concentration of heparin available to the cells.
Effects of growth factors on heparin-induced inhibition
As shown above, the addition of TGFe, bFGF, and aFGF resulted in overcoming inhibition of growth in soft agar. Similarly, monolayer growth inhibition by heparin could be overcome by bFGF, aFGF, and TGFe. This effect of all three growth factors was equivalent and more complete on day 7 than day 4. The growth factors were able to overcome heparin inhibition by
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nearly 100% in most assays performed on day 7 (Table 2). These results again suggest that the action of hep- arin is cytostatic rather than cytotoxic. The effect of EGF and IGF-I on growth of SW-13 cells in monolayer was examined as well. No effect of either growth factor in monolayer independent of the presence or absence of heparin was noted (Table 2). This corresponds well with our data indicating that EGF and insulin have no effect on the soft agar growth of SW-13 cells.
Effects of heparin on [3H]thymidine incorporation in SW-13 cells
Changes in [3H]thymidine incorporation in heparin- exposed SW-13 cells closely paralleled changes in cell numbers. When [3H]thymidine incorporation was fol- lowed in sparse cultures of SW-13 cells for several days after heparin addition to the culture medium, up to 54% inhibition was observed on day 6. The inhibition usually was first noticeable on day 3 or 4 and was max- imal on day 7 (Fig. 3). Both 24 hr and 1 hr pulses with 0.1 µCi/ml and 1.0 pCi/ml thymidine, respectively, showed the same degree of inhibition (data not shown).
HALPER AND CARTER
| Growth Factor | Day 4ª | Day 7ª | ||
|---|---|---|---|---|
| 0 µg/ml | 100 µg/ml | 0 µg/ml | 100 µg/ml | |
| Noneb | 100 | 52 ± 10 | 100 | 60 ± 21 |
| TGFe | ||||
| 3 U | 102 ± 16 | 76 ± 21 | 89 ± 8.4 | 102 ± 28 |
| 10 U | 86 ± 18 | 105 ± 25 | 99 ± 23 | 153 ± 56 |
| Noneb | 100 | 61 + 10 | 100 | 61 ± 25 |
| bFGF | ||||
| 1 ng | 83 ± 29 | 90 ± 11 | 117 ± 19 | 94 ± 25 |
| 10 ng | 76 ± 25 | 87 ± 4 | 122 ± 30 | 115 ± 34 |
| Nonec | 100 | 49 ± 6 | 100 | 74 ± 14 |
| aFGF | ||||
| 1 ng | 98 ± 13 | 71 ± 9 | 93 ± 10 | 111 ± 14 |
| 10 ng | 69 ± 6 | 72 ± 8 | 92 ± 6 | 123 ± 13 |
| Noneb | 100 | 69 ± 12 | 100 | 68 ± 13 |
| EGF 10 ng | 85 ± 12.5 | 79 ± 18 | 81 ± 9 | 62 ± 21 |
| IGF-I 10 ng | 86 ± 8 | 64 ± 7 | 82 ± 14 | 71 ± 13 |
SW-13 cells were inoculated in six well plates at b0.5 x 103 or “104 cells/well, heparin and growth factors were added 24 hr later, and cells were counted on days 4 and 7. Results are expressed as percent of number of cells in control wells + SD in six wells (TGFe, bFGF) or in quadruplicate (aFGF, EGF, IGF-I).
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Effects of heparin on protein synthesis
To study protein synthesis, exponentially growing cultures of SW-13 cells were transferred from medium with 5% CS and with or without 100 g/ml heparin into serum- and methionine-free DMEM with and without 100 µg/ml heparin and labeled with [35S]methionine for 3-4 hr. These studies were performed in replicate wells in parallel with [3H]thymidine incorporation studies and growth curves to determine whether any heparin-induced changes in protein synthesis corre- sponded to changes in cell numbers and [3H]thymidine incorporation. Cell-associated [35S]methionine uptake decreased consistently in cells exposed to heparin. However, these changes closely correlated with de- creased cell numbers and decreased [3H]thymidine incorporation in cells cultured with heparin. Auto- radiography of cell-associated proteins labeled with
[35S]methionine and separated on SDS-polyacrylamide gels revealed no quantitative or qualitative difference in specific proteins (data not shown). It thus appears that overall protein synthesis is unaffected by heparin.
The incorporation of [35S]methionine into TCA-pre- citable material was also diminished in the medium of SW-13 cells exposed to heparin, but to lesser degree than [35S]methionine uptake into the cells. However, preliminary data showed some quantitative changes in several specific proteins secreted into medium induced by heparin. These included a decrease in several pro- teins (60 and 25 K) and an increase in a 36 K protein. These changes are currently being investigated further in our laboratory.
Effects of heparin on [3H]thymidine incorporation in AKR-2B cells
We also studied the effects of heparin on mouse em- bryo AKR-2B cells. [3H]thymidine incorporation was followed in quiescent AKR-2B cells in serum-free me- dium. The [3H]thymidine incorporation assay in AKR- 2B cells was shown to correlate well with proliferation of these cells by other investigators (Shipley et al., 1984, 1985). Heparin alone further inhibited already quiescent AKR-2B cells by 50%. The moderate stimu- lation of AKR-2B cells caused by TGFe was decreased by 53% in the presence of 100 µg/ml heparin. However, already potent stimulation of AKR-2B cells by bFGF was further enhanced by heparin by 155% (Table 3).
DISCUSSION
Besides its activity as an anticoagulant, heparin has the ability to act as a growth modulator. It can inhibit or stimulate cell growth depending on cell type tested and growth factor(s) present. For example, heparin modulates the activity of bFGF and aFGF, especially potentiating the stimulatory activity of aFGF (Gimenez-Gallego et al., 1986; Gospodarowicz et al., 1986). Heparin was first shown to inhibit the prolifer- ation of vascular smooth muscle cells by Clowes and Karnovsky (1977).
Growth of several other cell types is inhibited by heparin as well. Paul et al. (1980) studied the inhibi-
| Growth factor | No heparin | 100 µg/ml Heparin |
|---|---|---|
| None | 1 | 0.5 ± 0.2 |
| 10 U TGFe | 9.1 ± 5.5 | 4.8 ± 1.8 |
| 10 ng bFGF | 52 ± 12 | 81 ± 28 |
1Quiescent AKR-2B cells in SF medium were exposed to heparin and growth factors for 22-23 hr. This was followed by 1 hr pulse with 1.0 uCi/ml [H]thymidine as described in Materials and Methods. The results are expressed as the ratio of the number of cells in wells with a growth factor or heparin to the number of cells in control plates ± SD. The cell number was determined in six wells for each data point.
tion of growth of 3T3 fibroblasts by heparin, and Hoover et al. (1980) showed that heparin inhibits the growth rat arterial smooth muscle cells as well as BHK fibroblasts. Mesangial cells are inhibited by heparin both in vivo (Coffey and Karnovsky, 1985) and in vitro (Castellot et al., 1985b). Two other epithelial cell types besides SW-13 cells have been shown to be sensitive to the inhibitory action of heparin: rat cervical epithelial cells (Wright et al., 1985; Wright and Karnovsky, 1987) and, only recently, human and mouse skin kera- tinocytes (Ristow and Messmer, 1988).
We have shown that heparin inhibits the growth of yet another epithelial cell line, human carcinoma SW- 13 cells, in soft agar and monolayer. In agreement with other studies (Wright et al., 1985; Fager et al., 1988), this inhibition is dose- and cell density-dependent, re- versible, and most effective on cells in exponentially growing monolayer cultures. It is interesting to note that the effects of heparin on SW-13 cells in soft agar are less dependent on heparin preparation and concen- tration than in monolayer, perhaps due to the low cell density used in soft agar growth assays or to intrinsic differences between anchorage-dependent and anchor- age-independent cell growth. It has been shown that heparin affects other epithelial cells, rat cervical epi- thelial cells, in their log phase (Wright et al., 1985), whereas smooth muscle cells appear to be most sensi- tive to heparin in the growth-arrested phase (Castellot et al., 1981; Reilly et al., 1986). As a result, in our study, both SW-13 cell number and [3H]thymidine in- corporation were decreased, whereas overall protein synthesis did not appear to be affected, as other studies also showed (Castellot et al., 1985a; Cochran et al., 1985). However, increase and decrease in specific pro- tein expression occurs (Majack and Bornstein, 1984; Majack et al., 1985; Castellot et al., 1985a; Cochran et al., 1985), corresponding to our preliminary data. Coch- ran et al. (1988) demonstrated that heparin modulates the secretion of three proteins in the 35,000-38,000 M, by vascular smooth muscle cells. Heparin increases the secretion of two of these proteins and decreases the excretion of the third one, which was identified as be- ing immunologically related to major excreted protein, a lysosomal proteinase. Studies dealing with these changes in SW-13 cells more specifically are currently underway in our laboratory.
In contrast to other investigators, we have shown that heparin-induced inhibition can be overcome by TGFe, bFGF, and aFGF, all growth factors binding to heparin with differing degrees of affinity. Both bFGF and aFGF bind tightly to heparin; they elute from hep- arin-Sepharose column with 2 M NaCl and 1.6 M NaCl,
respectively. TGFe can be eluted from heparin- Sepharose column with 0.5 M NaCl. TGFe is a 25 kD acid- and heat-stable polypeptide, which is unrelated to FGF and is present in neoplastic and nonneoplastic tissues; it stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer (Halper and Moses, 1983, 1987; Brown and Halper, 1988). The reversal of heparin inhibition by growth factors and the unchanged morphology of SW-13 cells exposed to heparin indicate that the action of heparin is cytostatic rather than cytotoxic. Reilly et al. (1987) successfully counteracted heparin inhibition of vascu- lar smooth muscle cells by EGF. The mechanism of EGF action is unknown; however, heparin (or heparan sulfate) diminishes EGF binding to EGF receptors in these cells (Reilly et al., 1987, 1988). Heparin inhibi- tion of RCEC can also be blocked by EGF but not by insulin (Wright et al., 1985; Wright and Karnovsky, 1987). In our study, neither EGF nor IGF-I exerted any significant effect on SW-13 cells in either monolayer or soft agar in the absence or presence of heparin. Fager et al. (1988) claim to reverse inhibitory effect of hepa- rin on human arterial smooth muscle cells with plate- let-derived growth factor (PDGF); however, they used FBS and human serum rather than purified PDGF. Reilly et al. (1986) and Benitz et al. (1986) were unable to reverse heparin inhibition of smooth muscle cell growth with PDGF.
We also tested mouse embryo AKR-2B cells for their responsiveness to heparin. These cells have fibroblastic morphology and are known to respond to several growth factors, including EGF and IGF-I (Shipley et al., 1984), TGFB (Moses et al., 1985), and TGFe (Halper and Moses, 1987; Brown and Halper, 1988). Thus hep- arin acts as a dual modulator of monolayer growth of AKR-2B cells depending on the presence or absence of other growth factors. Although heparin potentiated the already marked stimulation of AKR-2B cells by bFGF, it further inhibited already quiescent AKR-2B cells in the absence of any growth factors. Heparin also par- tially inhibited moderate stimulation of AKR-2B cells by TGFe (or TGFe partially reversed heparin-induced inhibition).
The mechanism by which heparin regulates growth is unknown. It probably protects aFGF and bFGF like- wise from heat and acid inactivation (Gospodarowicz and Cheng, 1986) and proteolysis in vitro (Rosengart et al., 1988), and, as already mentioned, it decreases EGF binding to EGF receptors. Flow cytometry analysis re- vealed that heparin arrests rat epithelial cervical cells in the G1 phase of the cell cycle (Wright et al., 1985) and smooth muscle cells in Go→ S transition or very early S phase (Castellot et al., 1985a).
The presence of negatively charged glycosaminogly- canes (GAGs), such as heparin and heparan sulfate, which are synthesized by smooth muscle cells (Fritze et al., 1985) and endothelial cells (Castellot et al., 1981) in the extracellular matrix, indicates that GAGs may play a role in the control of matrix metabolism and cell proliferation. Data from several studies indicate that GAGs regulate thrombospondin and fibronectin syn- thesis by vascular smooth muscle cells and their depo- sition into matrix (Majack et al., 1985; Lyons-Giordano et al., 1987). These GAGs appear, then, to inhibit the growth of vascular smooth muscle and epithelial cells,
a process likely to be important in wound repair. If impaired, it may contribute to unchecked growth, such as occurs in malignancy. In this respect, it is interest- ing to note that heparin inhibits soft agar growth of SW-13 cells. Soft agar (anchorage-independent) growth is considered to correlate well with tumorigenicity in vivo (Kahn and Shin, 1979). Several investigators have observed either tumor regression induced with heparin (Folkman et al., 1983; Coombe et al., 1987) or increased degradation of matrix related GAGs, such as heparan sulfate, by highly metastatic malignancies (Nakajima et al., 1983; Hennes et al., 1988).
The results of this and other studies support the hy- pothesis that heparin, TGFe, bFGF, and aFGF modu- late the growth of SW-13 cells and possibly of other epithelial cells in complex ways. Depending on the cell type involved and on the growth factors present, hep- arin also may function as a dual modulator of cell growth and proliferation.
ACKNOWLEDGMENTS
This project was supported by NIH grant CA44039 and by a grant from the Veterinary Medical Experi- ment Station, University of Georgia.
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