Sequential development of an angiogenic phenotype by ... - Nature

Oncogene (1997) 14, 1495 ± 1502  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Sequential development of an angiogenic phenotype by human ®broblasts progressing to tumorigenicity Olga V Volpert, Kristina M Dameron1 and NoeÈl Bouck Department of Microbiology-Immunology and R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, IL 60611, USA

As normal cells progress to malignancy they must acquire an angiogenic phenotype that will enable them to attract the blood vessels necessary to support their progressive growth. Here we de®ne the mechanism by which human ®broblasts cultured from Li Fraumeni patients and progressing to tumorigenicity in vitro become angiogenic. Initially cells were anti-angiogenic due to the secretion of high levels of inhibitory thrombospondin that overrode the modest amounts of the major inducer, vascular endothelial cell growth factor (VEGF), that were also produced. Cells became fully angiogenic in two steps, the ®rst dependent on the loss of both alleles of wild-type p53 which caused a drop of at least 20-fold in secreted thrombospondin and a fourfold increase in secreted VEGF. Angiogenic activity increased again upon transformation by activated ras due to a further twofold increase in VEGF. Changes in relative levels of VEGF mRNA were sucient to account for changes in secreted protein levels and in overall angiogenic activity. These studies demonstrate that an angiogenic phenotype able to support tumorigenicity can arise in a step-wise fashion in response to both oncogene activation and tumor suppressor gene loss and involve both a decrease in the secretion of inhibitors and the sequential ratcheting up of the secretion of inducers of angiogenesis. Keywords: neovascularization; ®brosarcoma; VEGF; thrombospondin; tumor progression

Introduction Angiogenesis, the formation of new capillaries from pre-existing ones, is crucial for tumor development (reviewed in Folkman, 1995). All successful tumors are angiogenic, attracting the nourishing new vasculature on which they depend by secreting a variety of substances that diuse to nearby endothelial cells and stimulate them to form new vessels. Angiogenic tumor cells arise during tumor progression from normal cells that are not angiogenic and are often anti-angiogenic in that they secrete high levels of molecules that inhibit neovascularization and render ineective the low levels of inducers of angiogenesis that are also produced (Bouck et al., 1996). As these normal cells progress to tumorigenicity, in addition to developing a variety of Correspondence: N Bouck 1 Current address: Department of Natural Sciences, Loyola University, Chicago, IL 60611, USA Received 9 September 1996; revised 22 November 1996; accepted 25 November 1996

cell autonomous traits that enable them to divide without constraint, they must also alter the mixture of molecules they secrete so that their eect on endothelial cells switches from anti-angiogenic to potently angiogenic. This change presumably involves both an increase in the secretion of inducers of angiogenesis and a decrease in elaboration of inhibitors, although this has never been directly demonstrated. The development of an angiogenic phenotype during tumor progression seems likely to be driven by the same oncogene activation and tumor suppressor gene loss that result in unregulated mitogenesis. Activated oncogenes can stimulate the angiogenic phenotype of the cell in a number of ways (Bouck et al., 1996). Some, like sis, encode inducers of angiogenesis, others increase the secretion of pro-angiogenic proteases or stimulate cells to elaborate angiogenic molecules. For example, production of the potent inducer VEGF (vascular endothelial growth factor) is stimulated by ras in NIH3T3 ®broblasts (Grugel et al., 1995) and in intestinal cell lines (Rak et al., 1995) where its continued secretion at high levels depends on the continued presence of the activated oncogene. Depending on the cell type, oncogenes occasionally contribute to the angiogenic phenotype by downregulating inhibitors of angiogenesis, as activated ras decreases levels of inhibitory thrombospondin in bronchial epithelial cells (Zabrenetsky et al., 1994). Loss of tumor suppressor genes usually contributes to a more angiogenic phenotype by decreasing the production of molecules that inhibit angiogenesis (Bouck et al., 1996). For example, loss of wt p53 results in dramatic decrease in the secretion of the inhibitor thrombospondin (TSP) in ®broblasts (Dameron et al., 1994a,b) and of an anonymous inhibitor in human glioblastoma cells (Van Meir et al., 1994). Tumor suppressors can also in¯uence the production of inducers. Although the relevance of these changes to the overall angiogenic phenotype of a cell have not yet been de®ned, VEGF is down-regulated by wt p53 in 293 cells (Mukhopadhyay et al., 1995) and by the VHL tumor suppressor gene in a renal cell carcinoma line (Seimeister et al., 1996) and possibly also in stromal cells of hemangiomas (Wizigmann-Voos et al., 1995). Development of an angiogenic phenotype can also be observed in vivo during tumor progression in mouse models and human tumors (Hanahan and Folkman, 1996). In some cases, including a transgenic mouse model in which expression of the bovine papilloma virus drives dermal cells to develop into ®brosarcomas, the switch from normal to very high vessel density around the tumor seems to occur in a single step and to correlate with the accumulation of multiple

Progression to an angiogenic phenotype OV Volpert et al

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molecular changes including increases in viral oncoproteins E5 and E6 and in cellular Jun-B and c-jun, loss of diploidy, mutation of p53 and the initiation of the release from the cells of angiogenic factor basic ®broblast growth factor (bFGF; Christofori and

Hanahan, 1994). In models involving other cell types such as keratinocytes developing into squamous cell carcinomas, the density of nearby vessels increases gradually during tumor progression (Hanahan and Folkman, 1996). Although the identity of the

Figure 1 Changes in angiogenic activity during human ®broblast progression to tumorigenicity. (a) Total stimulatory activity and total inhibitory activity present in media conditioned by ®broblasts at the indicated stages of tumor progression were determined from a series of dose/response curves, using as an assay the ability to induce or inhibit the migration of capillary endothelial cells. Units of inhibitory activity are reported as the reciprocal of the ED50 for inhibition of the migration of endothelial cells towards a standard inducer. Units of stimulatory activity are calculated as one third of the reciprocal of the ED50 for stimulation of endothelial cell migration and were measured in the presence of antibodies that inactivated inhibitory TSP. The one third correction re¯ects the observation that inhibitory media from early passage LF cells could be diluted over threefold and still retain its ability to abolish migration elicited by media conditioned by tumorigenic ras lines. Errors are derived from testing, where available, two to three separately derived individual clones. Normal, normal adult human ®broblasts; LF early, ®broblasts derived from Li Fraumeni patients at early passage before becoming immortal; LF late, ®broblasts from Li Fraumeni patients that had become immortal; Ras, Tu7, ras-transformed derivatives of late passage LF ®broblasts that were not tumorigenic; Ras, Tu+, rastransformed derivatives of late passage LF ®broblasts that were tumorigenic. (b ± d) Dose/response curves respresentative of those used to derive the activity curve in (a). Conditioned media were tested at increasing concentrations in the presence of antibodies that neutralized TSP. (b) Conditioned media from normal cells (^) and from LF early passage cells from strains MDAH041 (*) and 172 (!); (c), media from LF cells MDAH087 at early (*) and late (~) passages; (d), media from ras-transfected cells including non-tumorigenic rasD (!) and tumorigenic rasB (*)


vasoactive molecules that are secreted by developing tumor cells and involved in the evolution of an angiogenic phenotype in vivo can be inferred from staining and in situ hybridization, it has been dicult to determine if molecules detected in this way are major or minor players in the overall angiogenic phenotype of the developing tumor, and impossible to quantitate their individual contributions. To gain more insight into how normal human cells develop an angiogenic phenotype as they progress to malignancy, to identify major secreted angiogenic mediators and to link them with cancer genes that control their production, we have used a set of previously characterized human skin ®broblasts cultured from Li Fraumeni patients that carry one active and one mutant allele of the p53 tumor suppressor gene (Bischo et al., 1990, 1991). Unlike normal adult ®broblasts, these cells can spontaneously immortalize and then subsequently be driven on to tumorigenicity by the introduction of activated ras. Measuring total angiogenic activity of cells at various points during progression we were able to track their developing angiogenic phenotype, relate it to genetic changes and quantitate the molecules involved. These ®broblasts became angiogenic gradually in a step-wise fashion that depended on the loss of inhibitory TSP resulting from inactivation of wt p53 as well as on the ratcheting up of VEGF secretion, once as a result of p53 loss and again in response to activated ras.

Results Changes in net angiogenic activity during tumor progression For this work a series of previously described cell strains and cell lines were used that represent ®ve sequential stages in the in vitro progression of normal human ®broblasts to malignant cells able to form ®brosarcomas in nude mice: early passage adult ®broblasts cultured from a skin biopsy from a normal person (normal; containing two wild-type p53 alleles), early passage adult ®broblasts cultured from three Li Fraumeni patients (LF early; containing one mutant and one wild-type allele of p53); immortalized derivatives of these same three Li Fraumeni cultures (LF late; containing only mutant alleles of p53) and four ras transfected clones derived from one of the immortal Li Fraumeni lines, two of which were not tumorigenic in nude mice (RasD and E, Tu7) and two of which were tumorigenic (RasA and B, Tu+). To monitor the development of an angiogenic phenotype during this progression, changes in the net activity of both the positive and negative signals secreted by these cells were measured using a series of dose/response curves generated by capillary endothelial cell migration assays (examples in Figure 1b,c,d). There was no change in net inducing or inhibitory activity when normal ®broblasts were compared to ®broblasts cultured from Li Fraumeni patients that retained only a single allele of wild-type p53 (Figure 1a,b). As these Li Fraumeni ®broblasts progressed towards tumorigenicity, total secreted inhibitory activity dropped precipitously in a single step when the cells became immortal and remained low thereafter. Total stimula-

tory activity increased eight- to tenfold during progression in two steps, fourfold when cells became immortal (Figure 1a,c) and an additional twofold upon transformation with activated ras. As re¯ected in Figure 1, media conditioned by early passage Li Fraumeni ®broblasts were able to inhibit migration induced by media conditioned by ras-transformed tumorigenic cells when mixed at equal protein concentrations and when diluted as much as 1:3 (data not shown). Changes in inhibitory TSP It has been demonstrated previously that secretion of the inhibitor of angiogenesis, thrombospondin-1 (Good et al., 1990), is responsible for the inhibitory activity seen in media conditioned by early passage normal and Li Fraumeni ®broblasts and that TSP mRNA and protein levels fall dramatically upon immortalization (Dameron et al., 1994a,b). Quantitative Western analysis of media conditioned by ®broblasts at various stages of progression showed that secreted TSP protein levels were very high prior to immortalization, when it constituted up to 10% of total protein secreted by the cells (Figure 2). Secreted TSP levels plummeted thereafter, remaining at 0.6% of total protein or less as cells progressed on to tumorigenicity. Changes in stimulatory VEGF When inhibitory TSP was neutralized, inducing activity was revealed in media conditioned by early passage ®broblasts (Dameron et al., 1994a,b; Figure 3a). More than three fourths of this inducing activity, as measured by capillary endothelial cell migration, could be attributed to VEGF for it was inhibited by neutralizing anti-VEGF antibodies (Figure 3a). Neither TGF-b (transforming growth factor beta) nor bFGF (basic ®broblast growth factor) played a crucial role since their neutralizing antibodies were ineective at reducing migration (Figure 3a). At later stages in progression VEGF accounted for all measurable angiogenic activity in vitro as shown for representative clones (Figure 3b). In an attempt to detect other inducers which may be contributing via synergy with VEGF, media from LF late and RasB cells were tested in a migration assay with antibodies able to neutralize the angiogenic activity of aFGF, bFGF, PDGF, TGFb, IL-8 and IL-4. None of these antibodies decreased angiogenic activity. The dominance of VEGF demonstrated in the in vitro capillary endothelial cell migration assays was con®rmed using the in vivo rat corneal assay (Table 1). When measured by an ELISA assay, the total amount of bFGF secreted by these cells did increase during progression, from undetectable (51 pg/mg of secreted protein) in secretions of normal, LF early and LF late cells to 2 pg/mg secreted protein in ras transformed cells. Such low amounts do not register in angiogenesis assays and are inconsequential compared to the activity generated by VEGF. Re¯ecting the upregulation of net inducing activity (Figure 1a), quantities of VEGF made and secreted by ®broblasts in the course of tumor progression increased in two distinct steps. Relative VEGF mRNA levels measured by semi-quantitative RT ± PCR were very low in normal and LF early ®broblasts (Figure 4, lanes

1497


ha-TSP

RasD,Tu–

RasB,Tu+

Discussion RasA,Tu+

LF late (neo)

LF late

LF early

Normal

1498

TSP

TSP as a % of total secreted protein

10.4 9.6 0.01 0.55 0.02 0.05 0.03

Figure 2 Changes in secreted thrombospondin protein during tumor progression. Western blot showing levels of TSP in media conditioned by cells at various stages of progression. Notation as in legend to Figure 1 with letters indicating individual clones. LF late (neo) is a control clone of LF late cells trasnfected with neo. The proportion of total secreted protein that was determined to be TSP is indicated below each lane. Puri®ed hamster TSP (haTSP; Good et al., 1990) served as a positive control

Table 1

VEGF is a major inducer of neovascularization in early passage and tumorigenic fibroblasts

Test substance

Antibody added

Positive corneas/total

VEGF none 3/3 anti-VEGF none 0/2 VEGF anti-VEGF 0/2 LF early none 0/2 LF early anti-TSP 2/2 LF early + anti-TSP + anti-VEGF 0/3 RasA,Tu+ none 3/3 RasA,Tu anti-VEGF 0/3 Test substances were incorporated into pellets and implanted into the avascular rat cornea. Vigorous ingrowth of vessels towards the pellets by seven days was considered a positive response Controls

Conditioned media

5, 6) and increased 3.6-fold upon immortalization while secreted protein levels increased ®vefold (Figure 4, lanes 7, 8). This increase appeared to be due to the loss of the wild-type p53 gene that accompanies immortalization (Bischo et al., 1990, 1991), for shifts of similar magnitude in VEGF were seen when pools of late passage Li Fraumeni ®broblasts expressing temperature sensitive p53 grown at the high temperature, where p53 was mutant, were compared to the same cells grown at low temperature, where p53 was in the wild-type con®guration (Figure 4, lanes 3, 4), but not in neo-transfected controls (Figure 4, lanes 1, 2). An additional increase of about twofold in VEGF message occurred when immortal ®broblasts were transformed with activated Ha-ras (Figure 4). The increase in message of 2.3-fold was matched by the doubling in the amount of secreted protein and was sucient to account for the doubling of net angiogenic activity seen in Figure 1. No further changes in VEGF (Figure 4, lanes 9 ± 11) or in overall angiogenic activity (Figure 1a,d) was detected when tumorigenic and nontumorigenic clones of ras-transformed ®broblasts were compared.

Although it has been realized for some time that successful tumors must develop an angiogenic phenotype, data presented here detail for the ®rst time exactly how this process can happen. Using human ®broblasts progressing to malignancy in vitro as a model system it has been possible to identify TSP and VEGF as the major mediators of angiogenesis and p53 and ras as two genes able to modulate their secretion, and to show that quantitatively, loss of inhibitory TSP is crucial to development of an angiogenic phenotype sucient to support tumor growth in vivo. Normal human ®broblasts in culture were antiangiogenic, secreting high levels of inhibitory TSP and low levels of inducers, of which VEGF was the major one. The lack of neovascularization in normal tissues is usually attributed in theory to a balance between inducers and inhibitors that favors inhibitors (Bouck et al., 1996). The secretions of normal ®broblasts demonstrate such a balance for in this mix the activity of the VEGF and any other minor inducers secreted by the cells is counterbalanced by inhibitory TSP so that the overall activity is anti-angiogenic. These cells became angiogenic during tumor progression in a step-wise fashion. The ®rst step in tumor progression, the mutation of one of the two alleles of p53 had no discernible eect on the angiogenic phenotype. Despite the presence of mutant p53 protein with an arg to trp mutation at codon 248 in the Li Fraumeni early passage cells (Dameron et al., 1994a,b), no dominant negative eect on angiogenesis was seen. The loss of the remaining wild-type p53 allele was necessary to aect angiogenesis and resulted in an apparently simultaneous decrease in the secretion of inhibitory TSP and increase in secretion of inducing VEGF. As a result the net activity of cellular secretions switched from anti-angiogenic to angiogenic. It was the loss of inhibitory TSP that was the determining factor in this switch, for normal cells secreted three times the level of TSP sucient to inhibit angiogenic activity secreted by tumorigenic cells. Wild-type p53 supports the production of TSP at the RNA level (Dameron et al., 1994a,b) in part via a putative p53 response element in its ®rst intron (Stellmach and Bouck, unpublished data). The presence of wild-type p53 also depressed the basal level of VEGF message. A rise in VEGF mRNA levels upon loss of wild-type p53 has also been noted when mouse ®broblasts spontaneously immortalize and acquire a mutant p53 (Sugihara et al., 1994) and a similar regulation may underlie the ability of wt p53 to reduce basal levels of VEGF mRNA by one ®fth when introduced into 293 cells (Mukhopadhyay et al., 1995). Although wild-type p53 can inhibit a VEGF promoter construct (Mukhopadhyay et al., 1995), it is not yet clear if this is a direct or indirect eect. The second step in the development of the angiogenic phenotype occurred in response to the introduction of activated ras which resulted in a further rise in VEGF message and secretion. Activated ras induces a similar jump in VEGF mRNA in NIH3T3 ®broblasts (Grugel et al., 1995) and in human epithelial cells of colonic origin (Rak et al., 1995). It has been suggested that ras stimulates VEGF transcription via AP-1 sites in its promoter (Grugel et


1499

Figure 3 At all stages of progression VEGF was a major inducer secreted by the cells. (a) Media conditioned by early passage and late passage Li Fraumeni (LF) ®broblasts were assayed for ability to stimulate endothelial cell migration when tested alone (clear bars) or in the presence of antibodies that neutralize the inhibitory eects of TSP (shaded bars). To determine the molecules responsible for the inducing activity revealed when TSP was inactivated, additional monoclonals were added that neutralized either VEGF (a-VEGF), bFGF (a-bFGF) or TGF-b (a-TGF-b). In additional controls antibodies against TGF-b and against bFGF were neutral when tested alone, inhibited migration towards their respective targets [10 pg/ml TGF-b; 10 ng/ml bFGF], and did not interfere with migration towards VEGF. Dotted line indicates level of migration towards bFGF alone. (*)Indicates samples that diered signi®cantly from those tested with anti-TSP only (P50.003). (b) Media conditioned by late passage Li Fraumeni ®broblasts and by their derivatives that had been further transformed by ras were tested for ability to stimulate endothelial cell migration in the presence (shaded bars) and absence (clear bars) of neutralizing anti-VEGF antibodies. Dotted line indicates level of migration towards VEGF alone. (*)Indicates samples that diered signi®cantly from media controls (P50.002)

al., 1995), a hypothesis that is supported by in vivo experiments showing that high levels of VEGF and vascularization fail to develop in ras mediated skin tumors arising in fos knock out mice de®cient in AP-1 (Saez et al., 1995). The fact that less than half of the ras-transformed Li Fraumeni clones that have been isolated are tumorigenic (Bischo et al., 1991) suggests that further genetic changes in addition to ras activation are necessary in order for these cells to progress to full malignancy. This ®nal change did not augment the angiogenic activity of the cells. Thus the angiogenic phenotype developed in only two steps in response to p53 loss and ras activation was sucient to support the tumorigenicity of these cells. Despite the fact that there are a number of in¯uences on the angiogenic phenotype in vivo that

are missing in an in vitro system, including amplifying eects of hypoxia and accessory cells and the inhibitory in¯uences from surrounding normal cells, several lines of evidence suggest that the sequential changes described here in cultured ®broblasts accurately reflect changes taking place when ®brosarcomas develop naturally in vivo. In a transgenic mouse model of ®brosarcoma development, the switch to an angiogenic phenotype does coincide with the mutation of p53 (Christofori and Hanahan, 1994), as predicted from our human ®broblast progression in vitro. In these mice the switch also coincides with increases in JunB and cjun components of AP-1 and with the inauguration of the release of angiogenic bFGF from tumor cells, although it has not yet been possible to tell which of these events are essential to the angiogenic switch in


no cDNA

RasB, Tu+

RasA, Tu+

RasD, Tu–

LF late (neo)

LF late

LF early

neo p53 38° 32° 38° 32°

Normal

1500

VEGF

The variety of other oncogenes activated that substitute for ras in these tumors (Pollock et al., 1994) can also in¯uence angiogenesis (Bouck et al., 1996). Thus the in vitro data presented above may re¯ect with considerable accuracy events that happen in vivo as fibrosarcomas and other soft tissue sarcomas progress to malignancy. Materials and methods

1 Secreted VEGF

2

3

4

5

6

7

8

9 10 11 12

23 19 21 3.8 3.2 5.0 25 25 47 49 46 pg/µg total protein

Actin

1

2

3

4

5

6

7

8

9

10 11 12

Figure 4 VEGF mRNA and protein increased when wild-type p53 was inactivated and again upon introduction of activated ras. Lanes 1 ± 4. Southern blot of semi-quantitative RT ± PCR analysis of RNA obtained from late passage Li Fraumeni ®broblasts expressing either neo (lanes 1, 2) or a temperature sensitive p53 (lanes 3, 4) and grown at high or low temperature as indicated. Lanes 5 ± 12. Similar analysis of RNA from normal ®broblasts and from Li Fraumeni ®broblasts at various stages of progression towards tumorigenicity. LF late (neo) is a late passage Li Fraumeni line transfected with neo that serves as a control for clones transformed by ras. RNA samples were ampli®ed with primers for VEGF (top panel) or for actin (lower panel). Southern blots of ampli®ed DNAs are shown. The three VEGF bands detected correspond to mRNAs encoding VEGF isoforms of 189, 165 and 121 kDa. The amount of VEGF protein actually produced by each line in pg/mg of total secreted protein is indicated across the bottom

vivo. Although it was present at low levels in rastransformed ®broblasts, bFGF did not play a signi®cant role in the development of the angiogenic phenotype of these human ®broblasts progressing to tumorigenicity in vitro. On a molar basis it is much less active than VEGF and its concentration was below that necessary to induce migration or mitogenesis in vitro. In vivo simply neutralizing VEGF was sucient to remove all angiogenic activity from media conditioned by ras-transformed cells that contained measurable bFGF. The in vitro results reported here are also compatible with what is known about the development of human sarcomas in which loss of wild-type p53 function occurs with remarkably high frequency (Oliner et al., 1992; Leach et al., 1993; Andreassen et al., 1993) and is associated with poor outcome (Cardon-Cardo et al., 1994; Drobnjak et al., 1994), as might be expected if it mediated both the loss of inhibitory TSP and an increase in stimulatory VEGF. Although ras mutations are less common, they do occur in human rhabdomyosarcomas (Stratton et al., 1989) and ®brosarcomas (Tanaka et al., 1986; Maillet et al., 1992; Andeol et al., 1988) where mutant N-ras can be essential for the maintenance of transformation (Paterson et al., 1987).

Cell culture The normal human adult ®broblast strain MDAH170 derived from a skin biopsy (normal), Li Fraumeni early passage ®broblasts (LF early), MDAH087, MDAH041 and MDAH172 and their immortal late passage derivatives (LF late) and MDAH087 subclones transfected with neo or Haras were obtained from Dr M Tainsky (Bischo 1990, 1991). Whenever a single LF early or LF late sample is shown, these are derived from the MDAH087 lineage. Late passage MDAH041 expressing a val135 temperaturesensitive p53 mutant have been previously characterized (Dameron et al., 1994a). The ®broblasts were maintained in Dulbecco modi®ed Eagle medium (DME) supplemented with 10% fetal bovine serum and G418 where necessary. Early passage cells were used under passage 21, immortal cells at passages over 62. Bovine adrenal capillary endothelial cells BP10T8 were the kind gift of Dr J Folkman (Children's Hospital, Harvard Medical School), and were maintained in DME supplemented with 10% donor calf serum and 100 mg/ml endothelial cell mitogen (R&D Systems, Minneapolis, MN) and used at passage 15. Unless otherwise indicated, all the cells were incubated at 378C at 7% CO2. To collect conditioned media, cells that had reached 90% to 100% con¯uence were rinsed, incubated 4 ± 7 h in serumfree DME, rinsed extensively and incubated in serum-free DME for 48 ± 72 h. Conditioned media were collected, cleared of cell debris and concentrated 20 ± 50-fold with Amicon membranes with a 10 kDa molecular weight cut o. Total protein was determined using Bio-Rad protein assay kit (Bio-Rad Labs, Hercules, CA). Western analysis Ten mg of total protein from conditioned media was resuspended in loading buer containing 15% BME, boiled 3 ± 5 min, run on 6% SDS ± PAGE and blotted to HybondN membrane (Amersham, Arlington Heights, IL). The membrane was blocked overnight with 7% Blotto (Carnation non-fat dried milk in incomplete PBS) and probed with anti-thrombospondin monoclonal antibodies A4.1 (Good et al., 1990). After incubation for 2 h at room temperature the membrane was washed and secondary anti-mouse HRP-conjugated antibody (Sigma Corp, St Louis, MO) was added in 1% blotto. After 1 ± 2 h at room temperature the blot was developed with ECL kit (Amersham, Arlington Heights, IL) and exposed with Xray ®lm for 1 ± 20 s. TSP present in media conditioned by LF early ®broblasts was quantitated using densitometric analysis of Western blots comparing it to serial dilutions of puri®ed human TSP-1. Angiogenesis assays In vitro migration was assayed as previously described (Polverini et al., 1991). BP10T8 capillary endothelial cells were starved overnight in DME containing 0.1% bovine serum albumin, harvested, washed and suspended in DME with 0.1% BSA and plated at 26104 cells per well on the lower surface of a gelatinized polycarbonate membrane


(5 mM pore size; Nucleopore Corporation, Plesanton, CA) in an inverted modi®ed Boyden chamber. Cells were allowed to attach for 1.0 ± 1.5 h, then chambers were reinverted and test material added to the top well. Cells were further incubated at 378C, 7% CO2 for 3.5 ± 4.0 h. Membranes were ®xed, stained, mounted on slides and the number of cells that had migrated to the top side of the membrane per ten high power ®elds determined. DME with 0.1% BSA served as a negative control and recombinant human bFGF (10 ng/ml; R&D Systems, Minneapolis, MN) or VEGF (100 pg/ml; R&D Systems, Minneapolis, MN) were used as positive controls. To combine data from several experiments background was subtracted and data converted into percent maximum migration for each experiment. Negative values re¯ect migration levels below the baseline BSA control. To quantitatively assess total inducing or inhibitory activity, serial dilutions of conditioned media were assayed, a dose/response curve plotted, and the protein concentration resulting in 50% maximum response (ED50) determined using a regression line based on the linear portion of the curve. Inducing activity was measured in the presence of neutralizing anti-TSP antibodies to abolish possible masking eects of inhibitory TSP. To convert to activity units, reciprocal values of the ED50 were calculated and normalized using the maximal inducing activity exerted by tumorigenic ras-transformed cells as one unit. Neutralizing antibodies included polyclonal anti-human bFGF, PDGF, and IL-8 pan-speci®c polyclonal anti-TGF-b, polyclonal anti-human VEGF all from R&D Systems, (Minneapolis, MN) anti-IL-4, a gift from JR Tepper and monoclonal anti-TSP A4.1 (Good et al., 1990) puri®ed from ascites. Polyclonal antibodies were used at 5 ± 10 mg/ml, monoclonal at 20 ± 40 mg/ml. In vivo neovascularization was assessed in the rat cornea (Polverini et al., 1991). Brie¯y, a Hydron pellet of 3 ± 5 ml containing test substance was formulated and implanted in the cornea of anesthetized rat approximately 1.0 ± 1.5 mm from the limbus. Neovascularization was assessed 5 and 7 days after implantation. On day 7 animals were perfused with colloidal

carbon, eyes ®xed in 10% buered formalin, corneas excised, ¯attened and photographed for the record. Where indicated pellets contained conditioned media at 200 mg/ml, VEGF at 1 ng/ml anti-TSP antibody A4.1 at 40 mg/ml and/or antiVEGF polyclonal antibodies at 10 mg/ml. VEGF quanti®cation VEGF protein in the media was measured with an ELISA kit (R&D Systems, Minneapolis, MN). To assess message levels with RT ± PCR total RNA from cell lines was isolated in a single step with TRIZOL reagent (Gibco, BRL, Gaithesburg, MD) and 5 mg of each sample used for the 1st strand cDNA synthesis with SuperScript kit and MuMLV reverse transcriptase (Promega, Madison, WI). Serial dilutions of the reverse transcribed products were ampli®ed in a 15 cycle PCR reaction with actin primers 5'GGC ATC GTG ATG GAC TCC-3' (left) and 5'-GCT GGA AGG TGG ACA GCG A-3' (right), annealing temperature 608C. The dilutions yielding similar amounts of product were chosen for subsequent analysis. VEGF cDNA sequences were ampli®ed in 24 cycle reaction with primers 5'-caa aag ctt CGA CCA TGA ACT TTC TCG T3' (left, contains VEGF nucleotides 154 ± 170 and an added restriction site), and 5'-ccc aag ctt CAC CGC CTC GGC TTG TC-3' (right, contains VEGF nucleotides 787 ± 804) using an annealing temperature of 658C. Ampli®cation products were run on a 2.5% agarose gel, transferred to GeneScreen Nylon membrane (NEN, Boston, MA), hybridized with a VEGF probe (Keck et al., 1989), or a b-actin probe (Ponte et al., 1984) and quantitated by phosphorimager.

Acknowledgements The authors wish to thank F Bisho and M Tainsky for providing ®broblasts used in this work and Karyn Laursen for excellent technical help. This work was supported by NIH Grants CA52750 and CA64239.

References Andeol Y, Nardeux PC, Daya-Grosjean L, Brison O, Cerbian J and Suarez H. (1988). Intl. J. Cancer, 41, 732 ± 737. Andreassen A, Oyjord T, Hovig E, Holm R, Florenes VA, Nesland JM, Myklebost O, Hoie J, Bruland OS, Borrensen AL and Fodstad O. (1993). Cancer Res., 53, 468 ± 471. Bischo FZ, Strong LC, Yim SO, Pratt DR, Siciliano MJ, Giovanella BC and Tainsky MA. (1991). Oncogene, 6, 138 ± 186. Bischo FZ, Yim SO, Pathak S, Grant G, Siciliano MJ, Giovanella BC, Strong LC and Tainsky MA. (1990). Cancer Res., 50, 7979 ± 7984. Bouck N, Stellmach V and Hsu S. (1996). Adv. in Cancer Res., 69, 135 ± 174. Christofori G and Hanahan D. (1994). Seminars in Cancer Biol., 5, 3 ± 12. Cordon-Cardo C, Latres E, Drobnjak M, Oliva MR, Pollack D, Woodru JM, Marechal V, Chen J, Brennan MF and Levine AJ. (1994). Cancer Res., 54, 794 ± 799. Dameron KM, Volpert OV, Tainsky MA and Bouck N. (1994a). Cold Spring Harbor Symposia in Quant. Biol., LIX, 483 ± 489. Dameron KM, Volpert OV, Tainsky MA and Bouck N. (1994b). Science, 265, 1582 ± 1584. Drobnjak M, Latres E, Pollack D, Karpeh M, Dudas M, Woodru JM, Brennan MF and Cordon-Cardo C. (1994). J. Natl. Cancer Inst., 86, 549 ± 554.

Folkman J. (1995). Tumor angiogenesis. In: J Mendelshon, PM Howley, MA Israel and LA Liotta (eds.), The Molecular Basis of Cancer, pp. 206 ± 232. New York: W. B. Saunders. Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA and Bouck NP. (1990). Proc. Natl. Acad. Sci. USA, 87, 6624 ± 6628. Grugel S, Finkenzeller G, Weindel K, Barleon B and Marme D. (1995). J. Biol. Chem., 270, 25915 ± 25919. Hanahan D and Folkman J. (1996). Cell, 86, 353 ± 364. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J and Connolly D. (1989). Science, 246, 1309 ± 1312. Leach FS, Tokino T, Meltzer P, Burrell M, Oliner JD, Smith S, Hill DE, Sidransky D, Kinzler KW and Vogelstein B. (1993). Cancer Res., 53, 2231 ± 2234. Maillet MW, Robinson RA and Burgart LJ. (1992). Mod. Pathol., 5, 410 ± 414. Mukhopadhyay D, Tsiokas L and Sukhatme VP. (1995). Cancer Res., 55, 6161 ± 6165. Oliner JD, Kinzler KW, Meltzer PS, George DL and Vogelstein B. (1992). Nature, 358, 80 ± 83. Paterson H, Reeves B, Brown R, Hall A, Furth M, Bos J, Jones P and Marshall C. (1987). Cell, 51, 803 ± 812. Pollock RE. (1994). Sem. in Surgical Oncol., 10, 315 ± 322. Polverini PJ, Bouck NP and Rastinejad F. (1991). Methods in Enzymology, 198, 440 ± 450. Ponte P, Ng SY, Engel J, Gunning P and Kedes L. (1984). Nucleic Acids Res., 12, 1687 ± 1696.

1501


1502

Rak J, Mitsuhashi Y, Bayko L, Filmus J, Shirasawa S, Sasazuki T and Kerbel RS. (1995). Cancer Res., 55, 4575 ± 4580. Saez E, Rutberg SE, Mueller E, Oppenheim H, Smoluk J, Yuspa SH and Spiegelman BM. (1995). Cell, 82, 721 ± 732. Seimeister G, Weindel K, Mohrs K, Barleon B, MartinyBaron G and Marme D. (1996). Cancer Res., 56, 2299 ± 2301. Stratton MR, Fisher C, Gusterson BA and Cooper CS. (1989). Cancer Res. 49, 6324 ± 6327. Sugihara T, Kaul SC, Mitsui Y and Wadhwa R. (1994). Biochim. Biophys. Acta., 1224, 365 ± 370.

Tanaka T, Slamon DJ, Battifora H and Cline MJ. (1986). Cancer Res., 46, 1465 ± 1470. Van Meir EG, Polverini PJ, Chazin VR, Huang HS, de Tribolet N and Cavenee WK. (1994). Nature Genetics, 8, 171 ± 176. Wizigmann-Voos S, Breier G, Risau W and Plate KH. (1995). Cancer Res., 55, 1358 ± 1364. Zabrenetsky V, Harris CC, Steeg PS and Roberts DD. (1994). Int. J. Cancer, 59, 191 ± 195.