p73 overexpression increases VEGF and reduces thrombospondin-1 ...

Oncogene (2001) 20, 7293 ± 7300 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

ORIGINAL PAPERS

p73 overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis Faina Vikhanskaya1,4, Maria R Bani3,4, Patrizia Borsotti3, Carmen Ghilardi3, Roberta Ceruti2,3, Gabriele Ghisleni2, Mirko Marabese1, Raaella Giavazzi3, Massimo Broggini1 and Giulia Taraboletti*,3 1

Department of Oncology, Mario Negri Institute for Pharmacological Research, 20157 Milano, Italy; 2Istituto di Anatomia Patologica Veterinaria e Patologia Aviare, UniversitaÁ degli Studi, 20133, Milano, Italy; 3Department of Oncology, Mario Negri Institute for Pharmacological Research, 24125 Bergamo, Italy

Tumor neovascularization is controlled by a balance between positive and negative eectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These ®ndings suggest a potential role of p73 in tumor angiogenesis. Oncogene (2001) 20, 7293 ± 7300. Keywords: p73; angiogenesis; VEGF; ovarian carcinoma

thrombospondin-1;

Introduction Tumor angiogenesis, the formation of new vessels required for tumor growth and malignant behavior, is controlled by a precise balance of positive and negative eectors (Hanahan and Folkman, 1996). Tumor cells

*Correspondence: Giulia Taraboletti, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy; E-mail: [email protected] 4 The ®rst two authors contributed equally to this work Received 2 February 2001; revised 1 August 2001; accepted 7 August 2001

become angiogenic at early stages of tumor progression, and the switch to an angiogenic phenotype is considered a hallmark of malignancy. The angiogenic potential of tumors represents in part a response to environmental changes, mainly hypoxia. However, the angiogenic ability of tumor cells is often the direct consequence of genetic alterations. Oncogenes and tumor suppressor genes can directly modulate the expression of proangiogenic and antiangiogenic factors in an opposite way thus determining the overall angiogenic activity of tumors (Bouck, 1996). The production of VEGF and thombospondin-1 (TSP-1), two main regulators of angiogenesis, has been described to be controlled by oncogenes and tumor suppressor genes. VEGF, one of the fundamental positive eectors in tumor angiogenesis, is stimulated by mutated ras, v-src, c-fos and c-jun (Mukhopadhyay et al., 1995; Rak et al., 1995; Saez et al., 1995). The same genes exert an opposite eect on the production of TSP-1, a complex regulator of angiogenesis, whose loss has been associated with the malignant progression of tumors. Oncogenes such as v-myc (Tikhonenko et al., 1996), c-jun (Dejong et al., 1999; Mettouchi et al., 1994), v-src (Slack and Bornstein, 1994), and the polyoma virus middle T (Sheibani and Frazier, 1995) down-regulate the TSP-1 gene, while the oncosuppressor genes p53 (Dameron et al., 1994), Rb (Salnikow et al., 1994), PTEN (Wen et al., 2001), nm23 (Zabrenetzky et al., 1994) and genes on chromosome 10q (Hsu et al., 1996) stimulate it. The tumor suppressor gene p53 has been described as a critical determinant of the angiogenic potential of tumor cells. This gene is involved in the regulation of dierent cellular processes, including cell cycle control and apoptosis (Vogelstein et al., 2000). Through these controls, p53 exerts its activity as oncosuppressor, and indeed in many human tumors the gene is found mutated (Harris, 1996; Hollstein et al., 1997). In several tumor types, wt p53 has been shown to act as a negative controller of angiogenesis, as it downregulates the expression of the angiogenic factor VEGF (Mukhopadhyay et al., 1995) while stimulating the production of endogenous inhibitors of angiogenesis, including TSP-1 (Dameron et al., 1994) and the TSP

p73 as a regulator of tumor angiogenesis F Vikhanskaya et al

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related BAI1 (Nishimori et al., 1997). The loss of functional p53 results in high VEGF and low TSP-1 production, with consequent increase in angiogenic activity of the tumor (Volpert et al., 1997). Recently, other members of the p53 family have been cloned and characterized (Kaelin, 1999). P73, the ®rst relative identi®ed, shares high sequence homology with p53, particularly in the DNA-binding domain (Slack and Bornstein, 1994). However, in contrast to p53, analysis of p73 in dierent human tumors failed to detect any signi®cant mutation (Han et al., 1999; Nomoto et al., 1998; Takahashi et al., 1998). Rather, in many human tumors, wild-type p73 is overexpressed (Chi et al., 1999; Tokuchi et al., 1999; Yokomizo et al., 1999), a ®nding that questions the possible role of p73 as tumor suppressor. In previous works, we described that overexpression of p73 in a human ovarian cancer cell line expressing wild-type p53 resulted in alterations of normal p53 response and in modi®cation of the pattern of gene expression (Vikhanskaya et al., 2000, 2001). Recently, p73 has been shown to reduce the production of VEGF in Saos-2 osteosarcoma and A293 human fetal kidney cells, suggesting that p73, as well as p53, is involved in the regulation of tumor angiogenesis (Salimath et al., 2000). In the present work, to investigate the role of p73 in tumor angiogenesis, we have analysed the eect of p73 on the production of pro- and anti-angiogenic factors and on the overall angiogenic activity, by using two clones, that stably overexpress p73, derived from the human ovarian carcinoma cell line A2780.

Figure 1 Levels of TSP-1 in the serum-free conditioned medium of A2780/pCDNA3, A2780/p73.4 and A2780/p73.5. TSP-1 was measured with ELISA. Data, expressed as ng TSP-1/mg of protein in the supernatant, are the mean and s.e.mean of values obtained from 6 ± 8 dierent preparations of conditioned medium

Results The human ovarian carcinoma cell line A2780, expressing wild-type p53, was transfected with p73, and two clones, A2780/p73.4 and A2780/p73.5, that overexpressed p73 RNA and protein, were isolated (Vikhanskaya et al., 2000). We used this model to evaluate whether overexpression of p73 would aect the production of positive and negative eectors of angiogenesis. The levels of the antiangiogenic factor TSP-1 were measured by ELISA in serum free conditioned medium from A2780/pCDNA3 cells (transfected with the empty vector) and the two p73 overexpressing clones. Both A2780/p73.4 and A2780/p73.5 cells showed a dramatic decrease in TSP-1 secretion compared to control cells (Figure 1). In dierent preparations of the supernatant, the reduction in TSP-1 secretion ranged from 53 to 100% (mean 81%) for A2780/p73.4 (n=8) and from 98 to 100% for A2780/p73.5 (n=6). To investigate whether the decreased secretion of TSP-1 in the medium was due to a reduced synthesis of TSP-1, we measured by Northern blot and RT ± PCR the TSP-1 mRNA levels in parental and p73 overexpressing cells. In both clones the TSP-1 mRNA levels were almost undetectable by both RT ± PCR and Northern blot, while a clear band was observable in parental cells (Figure 2). To measure the consequences of p73 Oncogene

Figure 2 Northern blot (a) and RT ± PCR analysis (b) of TSP-1 mRNA levels in A2780/pCDNA3, A2780/p73.4 and A2780/p73.5 cells. (c) reports the 2033 bp TSP-1 promoter fragment activity in the three dierent cell lines. Values are the mean+s.d. of three independent experiments and are expressed as relative luciferase activity normalized by the renilla activity value

overexpression on the TSP-1 promoter activity, a 2033 bp fragment of the human TSP-1 promoter subcloned 5' to a reporter gene was transiently transfected in the three cell lines. TSP-1 promoter activity was lower in the two p73 overexpressing clones as compared to empty vector transfected cells (Figure 2). It has to be noted, however, that both clones still retained a signi®cant promoter activity, the reduction being 18% in A2780/p73.4 and 44% in A2780/p73.5. We next measured the levels of the angiogenic factor VEGF in the conditioned medium. By ELISA assay, we found that p73 overexpressing clones released 2.7-fold (A2780/p73.4) and twofold (A2780/p73.5) more VEGF


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Figure 3 Levels of VEGF in the conditioned medium of A2780/ pCDNA3, A2780/p73.4 and A2780/p73.5. VEGF was measured with ELISA. Data, expressed as pg VEGF/mg of protein in the supernatant, are the mean and s.d.mean of values obtained from two dierent preparations of conditioned medium

than control cells (Figure 3). The increased secretion of VEGF was associated with an increase in the VEGF mRNA levels, as measured by Northern blot (Figure 4). Analysis of the VEGF promoter activity in the three cell lines revealed a tendency to increase in p73 overexpressing clones. These dierences, however, were not statistically signi®cant (Figure 4). The half-life of VEGF mRNA was measured after incubation in medium containing actinomycin D both in parental and p73 overexpressing clones. The two clones showed a slight, but non-statistically dierent, increase in mRNA half-life that was found to be 80 and 85 min for A2780/ p73.4 and A2780/p73.5 cells, respectively, versus 78 min in A2780/pCDNA3 cells. In parallel we measured in all the three cell lines, the half-life of TSP-1 mRNA, that was approximately 130 min and similar in both parental and p73 overexpressing clones. To check whether the eects of p73 on the promoter of both VEGF and TSP-1 could be in¯uenced by the presence of a wt p53, we used an isogenic pair of cell lines diering for the expression of p53, i.e. the human colocarcinoma cell line HCT116, expressing a wt p53 and a subline derived from it in which p53 has been deleted by targeted homologous recombination. Transfection of VEGF and TSP-1 promoter construct in these cells resulted in a dierent basal level of luciferase. For VEGF, the basal luciferase activity, normalized by the renilla value, was higher in HCT1167/7 cells (60+7.1) compared to HCT116+/+ cells (25+4.8), while the opposite results were found for TSP-1 (22+3.0 and 40+3.7 in HCT1167/7 and HCT116+/+ cells, respectively). These ®ndings are in agreement with the reported role of p53 in regulating the expression of the two factors (Dameron et al., 1994; Mukhopadhyay et al., 1995). When we cotransfected the two promoter constructs together with p73, we found that in HCT1167/7 cells p73 induced

Figure 4 Northern blot analysis of VEGF mRNA (a) in A2780/ pCDNA3, A2780/p73.4 and A2780/p73.5 cells. (b) Reports the activity of the 1005 bp promoter fragment of VEGF in the parental and in the two p73 overexpressing clones. Values, expressed as relative luciferase activity normalized for the internal control renilla activity, are the mean+s.d. of three independent experiments

a decrease in VEGF promoter activity and an increase in TSP-1 promoter activity (Figure 5). In HCT116+/+ cells, the results were the opposite; p73 induced an increase in VEGF promoter activity and a slight decrease in TSP-1 promoter activity, in agreement with the data obtained in the wt p53 expressing A2780 cells. The eect of p73 on the expression of other angiogenic factors was also investigated by Northern blot analysis. The two p73 overexpressing clones showed an increased expression of the VEGF related PlGF and PDGF-B compared to control cells (Figure 6). Clone A2780/p73.5 also presented a strong increment in the 4 mRNA forms of FGF-2 mRNA (Figure 6). IL-8 was not expressed in both control and p73 overexpressing clones (not shown). We next investigated whether the increased production of angiogenic factors and the parallel reduction of the inhibitor TSP-1 would aect the angiogenic behavior of tumors. In vitro, we measured the ability of supernatants of the three cell lines to aect endothelial cell motility, a crucial event of the angiogenic process, known to be modulated by the factors examined in this study. Using the Boyden chamber assay, we found that the supernatants of the two p73 overexpressing clones were signi®cantly more active in stimulating the migration of endothelial cells than the supernatant of control cells (Table 1). To assess the eect of p73 on the in vivo angiogenic potential of tumor cells, both p73 overexpressing clones and empty vector transfected cells were implanted subcutaneously in nude mice. All the injected mice developed tumors, indicating that both control and p73 overexpressing cells were tumorigenic. Tumors with a diameter of approximately 7 mm (mean volume was 171.3+91.2 mm3) were harvested and tumor sections analysed for vessel density (Figure 7). Both A2780/p73.4 Oncogene


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Figure 6 Expression of angiogenic factors in A2780/pCDNA3, A2780/p73.4 and A2780/p73.5. Northern blot analysis of PlGF, FGF-2, PDGF-B and 18 s mRNA

Table 1 Chemotactic activity for endothelial cells of supernatants of p73-overexpressing clones Attractant

Number of migrated cells

RPMI A2780/pCDNA3 A2780/p73.4 A2780/p73.5

8.0+2.8 78.7+6.5 183.7+10.1** 119.7+8.5*

Serum free supernatant of the indicated cell line, or RPMI (baseline motility) was used as the attractant, in the lower compartment of Boyden chamber. The migration of bovine aortic endothelial cells, added to the upper compartment of the chamber, is expressed as the number of cells migrated in 10 high power ®elds (mean and s.d.mean). The results are from one experiment representative of two. **P50.001; *P50.005 (Student t-test)

Table 2 Evaluation of apoptosis in vitro and in vivo in p73 overexpressing clones Figure 5 TSP-1 and VEGF promoter activity in HCT116+/+ and HCT1167/7 cells after co-transfection with a p73 expressing vector. Values are reported as relative luciferase activity, with cells transfected with promoter constructs alone arbitrarily set as 1

and A2780/p73.5 tumors presented an increased number of vessels compared to controls. Mean vessel count per ®eld was 22.0+6.1 for A2780/pCDNA3 (n=4), 38.4+9.4 and 33.6+8.8 for A2780/p73.4 and A2780/ p73.5 cells, respectively (for both clones: P40.005 compared to A2780/pCDNA3, n=5). We also evaluated, both in vitro and in vivo, if the two p73 overexpressing clones had a dierent basal level of apoptosis. In vitro, by staining the nuclei with DAPI, we observed that the percentage of apoptotic Oncogene

Clone A2780/pCDNA3 A2780/p73.4 A2780/p73.5

Apoptosis in vitroa 2.4+1.2 4.6+2.9 9.9+2.9

Apoptosis in vivo TUNELb Active caspase -3c 2.8+3.4 9.2+4.3 30.6+7.9

3.8+1.7 5.0+6.7 10.8+5.3

a The percentage (mean and s.d.mean) of apoptotic cells in vitro, in exponentially growing cells, was evaluated by staining the nuclei with DAPI. bApoptosis in the in vivo tumor was evaluated by TUNEL assay. Results are expressed as number of apoptotic cells counted in 10 ®elds (mean and s.d.mean, n=5). cApoptosis in the in vivo tumor was evaluated by immunostaining for active caspase-3. Results are expressed as number of apoptotic cells counted in 10 ®elds (mean and s.d.mean, n=6).

cells in exponentially growing, p73 overexpressing clones was higher than in A2780/pCDNA3 (Table 2). In vivo, by both TUNEL assay and immunostaining of


Figure 7 CD-31 immunohistochemistry. Representative histochemical analysis of vascularization of tumors generated in vivo by A2780/pCDNA3 (a), A2780/p73.4 (b) and A2780/p73.5 (c) cells (806)

active caspase-3, we found that the two clones presented a higher number of apoptotic cells compared to the control cells (Table 2). Discussion This study shows that the expression of angiogenesis regulatory factors is modi®ed in p73 overexpressing ovarian carcinoma cells. By reducing the production of the inhibitor TSP-1 and increasing that of the angiogenic factor VEGF, as well as other angiogenic factors, p73 aects the overall angiogenic activity of these tumor cells. It is now known that oncosuppressor genes contribute to the maintenance of the non-angiogenic status of cells by stimulating the production of inhibitors of angiogenesis, while inhibiting that of angiogenic factors. The tumor suppressor gene p53, often taken as an exemplar prototype of these regulators, has been described as directly controling the expression of genes coding for mediators of angiogenesis, and its loss, causing an impaired balance of angiogenic and antiangiogenic factors, causes the angiogenic switch in several tumor types. In particular, p53 has been implicated in the control of TSP-1 expression in ®broblast and in some tumor types, including ®brosarcoma and bladder carcinoma (Dameron et al., 1994; Grossfeld et al., 1997; Holmgren et al., 1998). In human ovarian carcinoma cells, however, in preliminary studies we could not demonstrate a regulation of TSP-1 production by p53 (data not shown). Here we have studied the eect of p73, another member of the p53 family (Kaelin, 1999) on the production of angiogenesis regulatory factors by ovarian carcinoma cells. Although p73 is homologous to p53 and is able to transactivate p53 target genes (Kaghad et al., 1997; Levrero et al., 2000), it does not appear to act as a tumor suppressor gene, as the lack of p73 does not result in increased rate of spontaneous tumor formation in mice de®cient for p73 (Yang et al., 2000). We previously reported that p73 can eect the expression of p53 target genes (Vikhanskaya et al., 2000), it was therefore interesting to investigate the eect of p73 on the expression of p53-modulated angiogenesis regulators and ultimately on the acquisition of angiogenic potential by tumor cells.

P73 decreased the production of TSP-1 and increased that of VEGF, as well as of the other angiogenic factors P1GF, FGF-2 and PDGF-B. Both clones showed a general increase in the levels of proangiogenic factors although a slightly dierent pattern of expression was observed: VEGF levels were higher in clone A2780/p73.4 than in A2780/p73.5, in contrast, expression of FGF-2 was strongly increased in clone A2780/p73.5 but not in A2780/p73.4; P1GF and PDGF-B were elevated in both clones to a similar extent. For both TSP-1 and VEGF, an alteration in secreted protein and in steady state mRNA levels was observed. P73 showed only a partial eect on the activity of both TSP-1 and VEGF promoters, therefore indicating that the mechanism of p73 eect could be at least in part post-transcriptional (although for VEGF the half life was only marginally increased in the two p73 overexpressing clones, and for TSP-1 it did not change) or that other not yet de®ned regulatory sequences, not present in the promoter fragment analysed, could play a role in the regulation of these promoters. The increased production of VEGF is particularly signi®cant in ovarian carcinoma. This tumor has been reported to produce high amounts of VEGF, which, in turn, through its ability to increase vascular permeability, contributes to the formation of peritoneal ascites, often present in this neoplasm (Nagy et al., 1995). Although for other tumor types a direct autocrine activity of VEGF has been reported, the lack of VEGF receptor expression (KDR/¯k1 and ¯t-1) in A2780 parental and p73 overexpressing cells (not shown) excludes that this occurs in our system. The changes in the expression of modulators of angiogenesis induced by p73 in tumor cells did result in increased angiogenic activity in vitro, as shown by the increased chemotactic activity for endothelial cells. Moreover, tumors generated in vivo by p73 overexpressing cells showed a higher vascular density compared to controls. In contrast to what was expected, however, the increased vascularization did not confer a growth advantage to these tumors. This was probably due to the fact that, as we previously reported, overexpression of p73 induced the expression of proapoptotic genes in these cells (Vikhanskaya et al., 2000, 2001). Indeed, while the mitotic index was similar in tumors generated by cells transfected with empty vector or p73 cDNA (not shown), the rate of apoptosis was higher in p73 overexpressing clones, in agreement with the increased apoptosis of these cells in vitro. These ®ndings point to a complex role of p73 in tumor progression: the ®nal eect of p73 on tumor growth will depend on which of the two activities ± proangiogenic or pro-apoptotic ± predominates. Our ®ndings indicate that, in ovarian carcinoma cells, p73 overexpression increases the production of VEGF. It has been recently reported that, in transient transfection experiments, co-transfection of p73 and VEGF promoter results in transcriptional repression of the latter (Salimath et al., 2000). Similarly, in cotransfection experiments, we found that in SKOV-3

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ovarian carcinoma (p53 null), SW620 colon carcinoma cells (mutated p53), as well as in HCT-116 and in Saos2 cells (p53 null), p73 decreased VEGF promoter activity (data not shown), suggesting that the eect of p73 on VEGF expression might ultimately depend on the p53 status of the cells. In agreement, we have shown here that p73 has an opposite eect in cells expressing or not a wt p53, strongly suggesting that in cells expressing a wt p53, p73 could act as a competitor of p53 (Vikhanskaya et al., 2000). The data also suggest that p73 could exert a proangiogenic activity in cells with a functional p53. This is particularly important in the light of the discussed role of p73 in tumor progression. In dierent human tumors, including ovarian carcinomas, levels of p73 (mRNA and protein) are higher than in benign lesions (Chen et al., 2000; Chi et al., 1999; Ng et al., 2000; Tokuchi et al., 1999; Yokomizo et al., 1999; Zwahlen et al., 2000). These ®ndings suggest that overexpression of p73 in p53 expressing tumor cells might neutralize the antiangiogenic action of p53, therefore providing a mechanism, alternative to the loss of p53, for the acquisition of the angiogenic competence. Materials and methods Cells Clones derived from the human ovarian carcinoma cell lines A2780, A2780/pCDNA3 (transfected with empty vector), A2780/p73.4 and A2780/p73.5 (overexpressing p73a) were obtained and grown as previously reported (Vikhanskaya et al., 2000). HCT116 cells with p53 disrupted by targeted homologous recombination (clone 392.7, p537/7) and their relative controls (clone 40-16, p53+/+) were grown in Iscove's modi®ed Dulbecco's medium supplemented with 10% fetal calf serum. TSP-1 and VEGF secretion Subcon¯uent cultures of A2780/pCDNA3, A2780/p73.4 and A2780/p73.5 cells were washed three times with saline and incubated for 4 h with serum-free medium. The medium was discarded, fresh serum free RPMI (for TSP-1 ELISA and motility assay) or RPMI 1% FCS (for VEGF ELISA) was added and incubated for additional 24 h. The conditioned medium was then collected, centrifuged and frozen in aliquots. At least two independent preparations of conditioned medium were analysed. TSP-1 was measured with a two-antibody immunoassay. Plates (MaxiSorp Nunc-ImmunoPlates, Nunc, Denmark) were coated overnight at 48C with the monoclonal antibody against human TSP-1, ahTSP-1 (ATCC, HB 8432), then blocked with 3% BSA for 1 h at room temperature. After incubation with the supernatants or TSP standards (3 h at room temperature), polyclonal anti TSP antibodies (Taraboletti et al., 1990) were used as secondary antibody, followed by peroxidase-conjugated anti rabbit IgG (Sigma, St Louis, MO, USA) and 1,2-Phenylenediamine (Dako, Glostrup, Denmark) as chromogen. Human platelet TSP-1 (100 2.5 ng) was used as a standard. VEGF was measured with a QuantikineTM immunoassay kit (R&D system, Minneapolis, MN, USA) following the manufacturer's instructions.

Oncogene

Promoter activity Promoter regions of TSP-1 and VEGF, respectively 2033 bp and 1005 bp, subcloned 5' to the luciferase gene in the PGL2 basic vector (Promega, Italy), were used to analyse transcription control of these genes in the dierent clones. Cells were co-transfected with 4 mg of pGL2 derived plasmids containing the 5' ¯anking region of the TSP-1 and VEGF genes and 0.05 mg of pRL-SV40 used for internal normalization. Reporter gene activities were evaluated after 24 h using the Dual Luciferase system (Promega). Results are expressed as the percentage of the control luciferase activity normalized by the renilla activity value. The mean+s.d. of three independent experiments is shown. Co-transfection experiments in HCT116 cells were performed as described above with the addition of 10 micrograms of a human p73 expressing vector. Northern blot and RT ± PCR 15 mg total RNA from exponentially growing cells were electrophoresed through an agarose-formaldehyde denaturing gel and transferred onto nylon membranes. The ®lters were hybridized overnight at 428C with 32P-labeled cDNA dissolved in 50% formamide, 5% dextrane sulfate, 56SSPE (0.75 M NaCl, 0.05 M NaH2PO4, 5 mM EDTA pH 7.4), 16Denhardt's (Ficoll, polyvynilpirrolidone and BSA, 0.2 mg/ml each), 1% SDS, 100 mg/ml denatured salmon sperm. Filters were then washed twice at 658C with SSC 0.26 and SDS 0.1%. To account for the amount of RNA being analysed the ®lters were hybridized overnight at 558C with a 32P-end labeled oligonucleotide speci®c for 18 s rRNA and washed three times at 508C. For TSP-1 RT ± PCR, 1 mg of total mRNA was used. After reverse transcription to cDNA, 35 cycles of ampli®cation, each consisting of 1' at 958C, 1' at 558C and 1' at 728C were used using primers 5'-CGTCCTGTTCCTGATGCATG and 5'-GGCAGGACACCTTTTTGCAGA amplifying a fragment of 1000 bp (Bertin et al., 1997). The fragments were separated on agarose gels and visualized using ethidium bromide. TSP-1 and VEGF mRNA half-lives were measured in A2780/pCDNA3, A2780/p73.4 and A2780/p73.5 cells. Total RNA was extracted after 0, 20, 40, 60 and 120 min incubation in medium containing Actinomycin D (2 mg/ml). VEGF and TSP-1 mRNA levels were quantitated by Real time PCR using the SYBR Green RT ± PCR kit (Applied Biosystem). Beta actin was used as internal control. Evaluation of apoptosis in vitro Basal level of apoptosis in exponentially growing cells was assessed after staining cells with DAPI (4'-6'-diamino-2phenylindole). Cells were seeded on glass coverslips (25 000 cells/ml). Forty-eight hours after seeding, cells were ®xed in 70% ethanol, air-dried and stained with DAPI as previously reported (Debernardis et al., 1997). Apoptotic cells were independently counted by three operators and the mean+s.d. of six dierent counts, each consisting of at least 200 cells, were determined. In vivo staining of tumor vessels and apoptotic cells Female NCr-nu/nu mice were obtained from NCI-DTPFCRDC (Frederick, MD, USA) and used when 6 ± 8-weeksold. Mice were injected subcutaneously with 56106 cells. Growing tumors were harvested, snap frozen and 5 mm sections were cut with a cryostat and ®xed in cold acetone.


For immunohistochemical analysis of CD31 (a speci®c marker of endothelial cells), sections were immunostained with a rat anti-mouse CD31 monoclonal antibody (Vecchi et al., 1994), according to the described procedure (Taraboletti et al., 2000). Microvessel count was carried out on three ®elds (6250) chosen within the highest vascularized areas. Any endothelial cell or cluster of endothelial cells positive for CD31 was counted. For detection of apoptosis, sections were either subjected to TUNEL assay using the In Situ Cell Detection Kit (Boehringer Mannheim, Germany) or immunostained with a rabbit anti active caspase-3 (an early marker of apoptosis) monoclonal antibody (BD PharMingen, San Diego, CA, USA). For the latter, the procedure described in (Taraboletti et al., 2000) was used, except that the antibody was diluted 1 : 6000 and that sections were pre-incubated with goat serum. In both assays, count of apoptotic cells was carried out on 10 random ®elds (6400). Two independent observers obtained similar results. Chemotaxis assay The chemotactic response of bovine aortic endothelial cells to serum free supernatants of A2780/pCDNA3, A2780/p73.4

and A2780/p73.5 cells was carried out in Boyden chambers as previously described (Taraboletti et al., 1990).

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Acknowledgments We would like to thank GL Semenza (Baltimore, MD, USA) and P Bornstein (Seattle, WA, USA) for the VEGF and TSP-1 promoter constructs, B Vogelstein (Baltimore, MD) for HCT116+/+ and HCT116 7/7, A Vecchi (Milan, Italy) for the anti-CD31 antibody, M Martinelli for in vivo experiments, and E Scanziani (Milan, Italy) for helpful comments on the histochemical analysis of tumors. This work was partially supported by grants from the Italian Association for Cancer Research (AIRC), and the Italian Foundation for Cancer Research (FIRC). F Vikhanskaya is a visiting scientist from the Institute of Cytology (Russian Accademy of Sciences), St. Petersburg, Russia.

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