Human Melanoma Cells Secrete and Respond to Placenta Growth Factor and Vascular Endothelial Growth Factor
2000; Elsevier BV; Volume: 115; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2000.00199.x
ISSN1523-1747
AutoresPedro Miguel Lacal, Cristina Maria Failla, Elena Pagani, Teresa Odorisio, Cataldo Schietroma, Sabrina Falcinelli, Giovanna Zambruno, Stefania D’Atri,
Tópico(s)Lymphatic System and Diseases
ResumoThe vascular endothelial growth factor is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta growth factor, a member of the vascular endothelial growth factor family of cytokines, in tumor angiogenesis, even though placenta growth factor/vascular endothelial growth factor heterodimers have been recently isolated from tumor cells. To investigate the role of placenta growth factor and vascular endothelial growth factor homodimers and heterodimers in melanoma angiogenesis and growth, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta growth factor and vascular endothelial growth factor polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different vascular endothelial growth factor isoforms (VEGF121, VEGF165, and VEGF189) and transcripts for two placenta growth factor isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta growth factor expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for vascular endothelial growth factor and placenta growth factor receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic factors in melanoma growth. The vascular endothelial growth factor is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta growth factor, a member of the vascular endothelial growth factor family of cytokines, in tumor angiogenesis, even though placenta growth factor/vascular endothelial growth factor heterodimers have been recently isolated from tumor cells. To investigate the role of placenta growth factor and vascular endothelial growth factor homodimers and heterodimers in melanoma angiogenesis and growth, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta growth factor and vascular endothelial growth factor polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different vascular endothelial growth factor isoforms (VEGF121, VEGF165, and VEGF189) and transcripts for two placenta growth factor isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta growth factor expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for vascular endothelial growth factor and placenta growth factor receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic factors in melanoma growth. fms-like tyrosine kinase soluble fms-like tyrosine kinase insulin-transferrin-sodium selenite medium supplement kinase-insert-domain-containing receptor placenta growth factor vascular endothelial growth factor The growth of solid tumors and the development of metastases are greatly favored by the angiogenic process. Actually, the vascular density of a number of different types of primary tumors is predictive for their metastatic potential (Gasparini, 1997Gasparini G. Prognostic and predictive value of intra-tumoral microvessel densiy in human solid tumours.in: Bicknell R. Lewis C.E. Ferrara N. Tumour Angiogenesis. Oxford University Press, 2006: 29-45Google Scholar). Several factors produced by cancerous cells influence the angiogenic process (Folkman, 1995Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease.Nature Med. 1995; 1: 27-31Crossref PubMed Scopus (7013) Google Scholar). Among them, the vascular endothelial growth factor (VEGF) is produced by a variety of normal and neoplastic cells and acts as an endothelial-cell-specific mitogen and permeability factor (for review seeNeufeld et al., 1999Neufeld G. Cohen T. Gengrinovitch S. et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3051) Google Scholar). At least five VEGF species of 121 (VEGF121), 145 (VEGF145), 165 (VEGF165), 189 (VEGF189), and 206 (VEGF206) amino acids are generated by alternative mRNA splicing from a single gene (Neufeld et al., 1999Neufeld G. Cohen T. Gengrinovitch S. et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3051) Google Scholar) and characterized by different biologic properties (Cohen et al., 1995Cohen T. Gitay-Goren H. Sharon R. et al.VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells.J Biol Chem. 1995; 270: 11322-11326Crossref PubMed Scopus (237) Google Scholar;Poltorak et al., 1997Poltorak Z. Cohen T. Sivan R. et al.VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix.J Biol Chem. 1997; 272: 7151-7158Crossref PubMed Scopus (439) Google Scholar). Placenta growth factor (PlGF), a secreted dimeric protein of the VEGF family, shares a 53% sequence identity with the platelet-derived growth factor (PDGF)-like region of VEGF (Maglione et al., 1991Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Isolation of a human placenta cDNA coding for a protein related to vascular permeability factor.Proc Natl Acad Sci USA. 1991; 88: 9267-9271Crossref PubMed Scopus (809) Google Scholar). Similarly to VEGF, alternative splicing of PlGF mRNA produces at least three polypeptides of 149 (PlGF-1), 170 (PlGF-2), and 221 (PlGF-3) amino acids (Maglione et al., 1993Maglione D. Guerriero V. Viglietto G. et al.Two alternative mRN, As coding for the angiogenic factor placenta growth factor (PlGF), are transcribed from a single gene of chromosome, 14.Oncogene. 1993; 8: 925-931PubMed Google Scholar;Cao et al., 1997Cao Y. Ji W. Qi P. Rosin A. Cao Y. Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.Biochem Biophys Res Comm. 1997; 235: 493-498Crossref PubMed Scopus (151) Google Scholar). A highly basic 21 amino acid insertion in the carboxi-terminal region of PlGF-2 sequence results in the high heparin-binding affinity of this polypeptide, whereas neither PlGF-1 nor PlGF-3 bind heparin. PlGF has been detected in a limited number of tissues and cell types (Clark et al., 1998Clark D. Smith S. Licence D. et al.Comparison of expression patterns for placenta growth factor, vascular endothelial growth factor (VEGF),VEGF-B, VEGF-C., in the human placenta throughout gestation.J Endocrinol. 1998; 159: 459-467Crossref PubMed Scopus (161) Google Scholar) and its in vivo and in vitro angiogenic activity has been demonstrated only recently (Ziche et al., 1997Ziche M. Maglione D. Ribatti D. et al.Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic.Lab Invest. 1997; 76: 517-531PubMed Google Scholar). Naturally occurring VEGF/PlGF heterodimers have also been found in the supernatant of some tumor cell lines (DiSalvo et al., 1995DiSalvo J. Bayne M. Conn G. et al.Purification and characterization of a naturally occurring vascular endothelial growth factor/placenta growth factor heterodimer.J Biol Chem. 1995; 270: 7717-7723Crossref PubMed Scopus (238) Google Scholar;Cao et al., 1996bCao Y. Linden P. Shima D. et al.In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.J Clin Invest. 1996; 98: 2507-2511Crossref PubMed Scopus (179) Google Scholar). VEGF proangiogenic activity is mediated by two tyrosine kinase receptors present on endothelial cells: the 180kDa fms-like tyrosine kinase (Flt-1) and the 200kDa kinase-insert-domain-containing receptor (KDR) (Neufeld et al., 1999Neufeld G. Cohen T. Gengrinovitch S. et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3051) Google Scholar). The binding of VEGF to KDR has been correlated with endothelial cell proliferation (Millauer et al., 1993Millauer B. 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Breier G. et al.The vascular endothelial growth factor receptor Flt-1 mediates biological activities.J Biol Chem. 1996; 271: 17629-17634Crossref PubMed Scopus (744) Google Scholar). The role of Flt-1 in other biologic activities triggered by VEGF is still controversial. A soluble form of Flt-1 (sFlt-1), lacking part of the extracellular domain, the transmembrane, and intracellular regions (Kendall and Thomas, 1993Kendall R.L. Thomas K.A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.Proc Natl Acad Sci USA. 1993; 90: 10705-10709Crossref PubMed Scopus (1128) Google Scholar), has been cloned. sFlt-1 binds VEGF and inhibits its mitogenic activity in vascular endothelial cells by impairing VEGF binding to KDR (Kendall and Thomas, 1993Kendall R.L. Thomas K.A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.Proc Natl Acad Sci USA. 1993; 90: 10705-10709Crossref PubMed Scopus (1128) Google Scholar). PlGF shares with VEGF the capability to bind with high affinity the Flt-1 receptor (Sawano et al., 1996Sawano A. Takahashi T. Yamaguchi S. Aonuma M. Shibuya M. Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta gowth factor, which is related to vascular endothelial growth factor.Cell Growth Differ. 1996; 7: 213-221PubMed Google Scholar), whereas PlGF/VEGF heterodimers are also able to activate KDR (DiSalvo et al., 1995DiSalvo J. Bayne M. Conn G. et al.Purification and characterization of a naturally occurring vascular endothelial growth factor/placenta growth factor heterodimer.J Biol Chem. 1995; 270: 7717-7723Crossref PubMed Scopus (238) Google Scholar;Cao et al., 1996bCao Y. Linden P. Shima D. et al.In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.J Clin Invest. 1996; 98: 2507-2511Crossref PubMed Scopus (179) Google Scholar). More recently, VEGF165 and PlGF-2 have been shown to bind also to neuropilin-1 and neuropilin-2, which function as receptors for axon guidance factors of the semaphorin family (Soker et al., 1998Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1997) Google Scholar;Gluzman-Poltorak et al., 2000Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. Neuropilin-2 and neuropilin-1 are receptors for VEGF165 and PlGF-2, but only neuropilin-2 functions as a receptor for VEGF145.J Biol Chem. 2000; 275: 18040-18045Crossref PubMed Scopus (294) Google Scholar). Nevertheless, both neuropilin receptors lack tyrosine kinase activity, and binding of VEGF165 to porcine aortic endothelial cells expressing only neuropilin-1 or neuropilin-2 did not affect cell proliferation or migration (Soker et al., 1998Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1997) Google Scholar;Gluzman-Poltorak et al., 2000Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. Neuropilin-2 and neuropilin-1 are receptors for VEGF165 and PlGF-2, but only neuropilin-2 functions as a receptor for VEGF145.J Biol Chem. 2000; 275: 18040-18045Crossref PubMed Scopus (294) Google Scholar). The role of VEGF in the process of tumor-induced angiogenesis is well established (reviewed byFerrara, 1995Ferrara N. The role of vascular endothelial growth factor in pathological angiogenesis.Breast Cancer Res Treat. 1995; 36: 127-137Crossref PubMed Scopus (403) Google Scholar), and several studies have focused on the intriguing possibility that VEGF could also directly affect tumor cell proliferation. VEGF tyrosine kinase receptors (Flt-1 and KDR) have been found in several human tumor cells, such as ovarian carcinoma (Boocock et al., 1995Boocock C.A. Charnock-Jones D.S. Sharkey A.M. et al.Expression of vascular endothelial growth factor and its receptors flt-1 and KDR in ovarian carcinoma.J Natl Cancer Inst. 1995; 87: 506-516Crossref PubMed Scopus (452) Google Scholar), AIDS-Kaposi sarcoma (Masood et al., 1997Masood R. Cai J. Zheng T. et al.Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (255) Google Scholar), prostate carcinoma (Ferrer et al., 1999Ferrer F.A. Miller L.J. Lindquist R. et al.Expression of vascular endothelial growth factor receptors in human prostate cancer.Urology. 1999; 54: 567-572Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), colon carcinoma (André et al., 2000André T. Kotelevets L. Vaillant J.-C. et al.VEGF, VEGF-B, VEGF-C and their receptors KDR, Flt-1 and Flt-4 during the neoplastic progression of human colonic mucosa.Int J Cancer. 2000; 86: 174-181Crossref PubMed Scopus (141) Google Scholar), and melanoma (Gitay-Goren et al., 1993Gitay-Goren H. Halaban R. Neufeld G. Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors.Biochem Biophys Res Comm. 1993; 190: 702-709Crossref PubMed Scopus (135) Google Scholar;Graeven et al., 1999Graeven U. Fiedler W. Karpinski S. et al.Melanoma-associated expression of vascular endothelial growth factor and its receptors Flt-1 and KDR.J Cancer Res Clin Oncol. 1999; 125: 621-629Crossref PubMed Scopus (78) Google Scholar). In a melanoma cell line, the expression of KDR receptor has been correlated with a proliferative response to exogenously added VEGF (Liu et al., 1995Liu B. Earl H.M. Baban D. et al.Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF.Biochem Biophys Res Commun. 1995; 217: 721-727Crossref PubMed Scopus (128) Google Scholar). In this report, however, the role played by the other VEGF receptors as well as the production of PlGF by melanoma cells were not analyzed. We were interested in studying the possible role of PlGF in melanoma angiogenesis and progression. To this end, we have characterized a wide variety of primitive and metastatic human melanoma cell lines for the expression of VEGF, PlGF, and their receptors, analyzed the in vivo expression of PlGF in cutaneous melanomas, and evaluated the effect of exogenously added VEGF and PlGF on melanoma cell proliferation. Culture media and supplements were purchased from Euroclone (Oud-Beijerland, Holland), except for the 0.85g per liter NaHCO3/minimum essential medium to grow WM115 cells, which was obtained from Gibco/BRL (Paisley, U.K.). Insulin-transferrin-sodium selenite medium supplement (ITS) and heparin were from Sigma (St. Louis, MO). Fatty-acid-free bovine serum albumin (BSA) was from Boehringer Mannheim (Mannheim, Germany). VEGF and PlGF homodimers and heterodimers used as standards in the enzyme-linked immunosorbent assay (ELISA) and as stimuli in the mitogenic assays were from R&D Systems (Abingdon, U.K.); rhVEGF165 homodimer was expressed and purified from Sf21 insect cells; PlGF-1 was expressed in Escherichia coli; VEGF/PlGF heterodimer was originated by expression in E. coli of VEGF165 and PlGF-1, followed by in vitro dimerization. Goat antibodies used in the ELISA analysis (R&D Systems) recognized all the VEGF or PlGF isoforms. The monoclonal antibodies used to neutralize VEGF (MAB 293) or PlGF (MAB 264) stimulated cell growth were also from R&D Systems. The goat polyclonal antibody raised against human PlGF and utilized for the immunohistochemical analysis was from Santa Cruz Biotechnology (Santa Cruz, CA). Nineteen human melanoma cell lines of primary or metastatic origin were used. Six were established in our laboratory (GR-Mel, ST-Mel, SN-Mel, PR-Mel, CN-Mel, and TVMBO). SK-Mel-28 (Shiku et al., 1976Shiku H. Takahashi T. Oettgen H.F. Old L.J. Cell surface antigens of human malignant melanoma. II. Serological typing with immune adherence assays and definition of two new surface antigens.J Exp Med. 1976; 144: 873-881Crossref PubMed Scopus (169) Google Scholar), WM115, and WM266-4 (Satyamoorthy et al., 1997Satyamoorthy K. De Jesus E. Linnenbach A.J. Melanoma cell lines from different stages of progression and their biological and molecular analysis.Mel Res. 1997; 7: S35-S42Crossref PubMed Scopus (133) Google Scholar) were purchased from the American Type Culture Collection (ATCC, Rockville, MD), and the remaining cell lines were generous gifts from other laboratories: 13443-Mel (Colombo et al., 1992Colombo M.P. Maccalli C. Mattei S. et al.Expression of cytokine genes, including IL-6, in human malignant melanoma cell lines.Mel Res. 1992; 2: 181-189Crossref PubMed Scopus (93) Google Scholar) from Dr. G. Parmiani (Istituto Nazionale Tumori, Milan, Italy); PD-Mel, PNP-Mel, PNM-Mel, LCP-Mel, LCM-Mel, and GL-Mel from Dr. F. Guadagni (Istituto Regina Elena, Rome, Italy); M14 (Golub et al., 1976Golub S.H. Hanson D.C. Sulit H.L. et al.Comparison of histocompatibility antigens on cultured human tumor cells and fibroblasts by quantitative antibody absorption and sensitivity to cell-mediated cytotoxicity.J Natl Cancer Inst. 1976; 56: 167-170PubMed Google Scholar) from Dr. G. Zupi (Istituto Regina Elena); LB-24 (Wolfel et al., 1993Wolfel T. Hauer M. Klehmann E. Analysis of antigens recognized on human melanoma cells by A2-restricted cytolytic T lymphocytes (CTL).Int J Cancer. 1993; 55: 237-244Crossref PubMed Scopus (90) Google Scholar) and 397-Mel (Topalian et al., 1989Topalian S.L. Solomon D. Rosenberg S.A. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas.J Immunol. 1989; 142: 3714-3725PubMed Google Scholar) from Dr. T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium). Melanoma cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine, and 50μg perml gentamycin, except for the ATCC cell lines WM115 and WM266-4, which were kept in culture as suggested by the supplier. Five of these cell lines were obtained from primary tumors (GR-Mel, ST-Mel, WM115, LCP-Mel, and PNP-Mel), six from cutaneous metastases (SN-Mel, PR-Mel, M14, 397-Mel, LB-24, and WM266–4), and eight originated from noncutaneous metastases, including lymph nodal and organ tissue metastases (CN-Mel, PD-Mel, 13443-Mel, PNM-Mel, LCM-Mel, GL-Mel, SK-Mel-28, and TVMBO). Normal human melanocytes were isolated from human skin biopsies at the IDI-IRCCS laboratory of Dr. M. De Luca (Pomezia, Italy) and in our own laboratory, as previously described (De Luca et al., 1988De Luca M. D'Anna F. Bondanza S. Franzi A.T. Cancedda R. Human epithelial cells induce human melanocyte growth in vitro but only skin keratynocytes regulate its proper differentiation in the absence of dermis.J Cell Biol. 1988; 107: 1919-1926Crossref PubMed Scopus (128) Google Scholar), NIH/3T3 murine fibroblasts and RAJI (human Burkitt's lymphoma) cells were purchased from the ATCC, and the human microvascular endothelial cell line HMEC-1 was a generous gift of Dr. F.J. Candal (Centers of Disease Control and Prevention, Atlanta, GA) (Ades et al., 1992Ades E.W. Candal F.J. Swerlick R.A. et al.HMEC-1: establishment of an immortalized human microvascular endothelial cell line.J Invest Dermatol. 1992; 99: 683-690Abstract Full Text PDF PubMed Google Scholar). Normal human keratinocytes were obtained from foreskins of healthy males, and cultured on a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (a gift from Dr. H. Green, Harvard Medical School, Boston, MA), as previously described (Zambruno et al., 1995Zambruno G. Marchisio P. Marconi A. et al.Transforming growth factor-β1 modulates β1 and β5 integrin receptors and induces de novo expression of the αvβ6 heterodimer in normal human keratinocytes: implications for wound healing.J Cell Biol. 1995; 129: 853-865Crossref PubMed Scopus (305) Google Scholar). Conditioned media from human melanoma cells and normal human melanocytes in culture were obtained by incubating semiconfluent cell cultures for 24h in 0.1% BSA/RPMI medium without fetal bovine serum. These conditions did not significantly affect cell viability, and the percentage of adherent cells was maintained between 93% and 99%. Supernatants were concentrated at least 10-fold in Centriplus concentrators (Amicon, Beverly, MA). Cells were detached from the flasks with phosphate-buffered saline (PBS)/ethylenediamine tetraacetic acid (EDTA) or, when necessary, with trypsin/EDTA. Cytokine secretion values were normalized by the total number of cells. To quantify unreleased VEGF or PlGF, cell extracts were prepared by washing cells once in PBS and resuspending them in lysis buffer (20mM Tris-HCl, 150mM NaCl, 1mM AEBSF, 1% Nonidet P-40, pH8.0). After 30min of incubation on ice, cell lysates were centrifuged and supernatants were directly used for ELISA analysis. Quantification of the amount of VEGF and PlGF homodimers and heterodimers in the conditioned medium and cell extracts was performed as previously described (Harlow and Lane, 1988Harlow E. Lane D. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) using goat anti-VEGF or anti-PlGF IgGs (R&D Systems) at a concentration of 10μg perml in PBS to coat Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark). Detection of the cytokines was performed with biotinylated goat anti-VEGF or anti-PlGF IgGs (R&D Systems) and streptavidin-alkaline phosphatase conjugate (1:10,000) (Boehringer Mannheim). For the detection of VEGF/PlGF heterodimers, plates were coated with anti-VEGF antibody and detection was carried out with biotinylated goat anti-PlGF IgGs. The reaction was stopped and optical density at 405nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA). This assay allowed detection of VEGF and PlGF polypeptides at concentrations equal to or greater than 100pg perml. Sections of melanoma specimens 4μm thick were deparaffinized, rehydrated, and treated for 20min with 0.3% H2O2 in PBS. Sections were preincubated with 3% BSA/PBS for 1h, and then incubated with the C-20 anti-PlGF antibody (Santa Cruz Biotechnology) at a concentration of 4μg perml for 2h at room temperature in 3% BSA/PBS. Specimens were subsequently treated for 45min with biotinylated antigoat IgGs (1:150) (Vector Laboratories, Burlingame, CA) diluted in 3% BSA/PBS, and with peroxidase-conjugated avidin (Vectastain Elite ABC kit, Vector Laboratories) for 1h. Immunoreactivity was visualized by peroxidase reaction, using 3-amino-9-ethyl carbazole (AEC, Vector Laboratories) in H2O2 as substrate, and specimens were counterstained with hematoxylin. Negative controls were obtained by omitting the primary antibody. In a previous analysis on PlGF expression during wound healing (Failla et al., 2000Failla C. Odorisio T. Cianfarani F. et al.Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000Abstract Full Text Full Text PDF Scopus (88) Google Scholar) we demonstrated the suitability of the Santa Cruz polyclonal antibody sc-1880 (C-20) for immunohistochemistry, checking its specificity by Western blotting against human recombinant VEGF and PlGF, and confirming the data by in situ hybridization analysis. The study was approved by the Ethical Committee of the Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy. Total cellular RNA from the different cell lines and cultured normal human cells (melanocytes and keratinocytes) was prepared using an RNeasy Midi kit from Qiagen (Hilden, Germany), following the manufacturer's directions. Three micrograms of total RNA per sample were used for reverse transcription by the AMV enzyme (Boehringer Mannheim), for 60min at 42°C, in 25μl. Five microliters of this cDNA preparation were used for each PCR amplification reaction by 1U of Dynazyme II DNA polymerase (Finnzymes OY, Espoo, Finland), utilizing the following primers and annealing conditions: forward primer 5′-CCATGAACTTTCCTG- CTGTCTT-3′ and reverse primer 5′-TCACCGCCTCGGCTTGTC-3′ for VEGF (annealing for 30s at 60°C); forward primer 5′-CTCCTA- AAGATCCGTTCTGG-3′ and reverse primer 5′-GGTAATAAATAC- ACGAGCCG-3′ for PlGF (annealing for 30s at 55°C); forward primer 5′-CACAGGAAACCTGGAGAATCAGACGACAAG-3′ and reverse primer 5′-TGGTCGACCATGACGATGGACAAGTA-3′ for KDR (annealing for 1min at 58°C); forward primer 5′-GAAGGAAGGGAG- CTCGTCATTC-3′ and reverse primer 5′-TACCATATGCGGTACAA- GTCAGG-3′ for the extracellular Flt-1 region (annealing for 1min at 60°C); forward primer 5′-CAAGTGGCCAGAGGCATGGAGTT-3′ and reverse primer 5′-GATGTAGTCTTTACCATCCTGTTG-3′ for the Flt-1 carboxi-terminal end (annealing for 1min at 60°C); forward primer 5′-ATGGAGAGGGGGCTGCCG-3′ and reverse primer 5′-CTATCGCG- CTGTCGGTGTA-3′ for neuropilin-1 (annealing for 30s at 52°C); forward primer 5′-ATGGATATGTTTCCTCTCACC-3′ and reverse primer 5′-GTCCAGCCAATCGTACTTGC-3′ for neuropilin-2 (annealing for 30s at 55°C). RNA integrity and the correct reverse transcription of the samples were assessed by testing each cDNA preparation for the amplification of the housekeeping gene glyceraldehyde-phosphate dehydrogenase (GAPDH). Controls were performed excluding AMV reverse transcriptase from the reactions. Amplification products obtained after RT-PCR were purified using the QIAquick gel extraction kit (Qiagen), and sequenced using the dye terminator cycle sequencing kit, in an Applied Biosystems PRISM 377 DNA Sequencer (Perkin Elmer, Foster City, CA). The capability of VEGF and PlGF homodimers and heterodimers to stimulate human melanoma cell growth was tested by measuring cellular methyl-3[H]-thymidine uptake or by counting the number of cells following incubation with these growth factors. For the methyl-3[H]-thymidine uptake experiments, cells were seeded on 96-well plates (3 × 103-5×103 cells per well, depending on the cell line), in 100μl of complete RPMI medium, and kept overnight at 37°C in a CO2 incubator. Subsequently, medium was changed with serum-free RPMI containing 0.1% BSA and cells were maintained at 37°C for two further days. Medium was then replaced with RPMI containing 0.1% BSA, 1μg perml heparin, and one of the growth factors or the medium supplement ITS, in quadruplicate samples. Samples with only BSA/heparin medium were included to determine basal radioactivity uptake. After 15h incubation, 1μCi of methyl-3[H]-thymidine (73Ci per mmol, Amersham, Buckinghamshire, U.K.) was added to each well, and the plates were further incubated for 5h. Labeled cells were transferred to Unifilter GF/C microplates (Canberra Packard, Meriden, CT) and the excess of nonincorporated thymidine was washed out, using a Filtermate 196-cell harvester (Canberra Packard). Radioactivity retained in the filter plates was then counted in a Top-Count microplate scintillation counter (Canberra Packard). The specific inhibition of VEGF- or PlGF-induced methyl-3[H]-thymidine uptake was performed by preincubating these cytokines or ITS (as negative control) with the respective monoclonal antibodies, for 1h at room temperature. Preincubation of the cytokines with a control monoclonal antibody (anti-HLA DP from Becton & Dickinson, San Jose, CA) was carried out simultaneously. For direct cell number counting, cells were seeded in 24-well plates (2×104 cells per well), allowed to attach to the plastic overnight, and serum-starved in 0.1% BSA/RPMI for 24h before stimulation. Stimuli (10ng perml of the cytokines and 5μg perml ITS as control) were added in the presence of 1μg perml heparin in 0.1% BSA/RPMI. Seventy-two hours after the addition of the different cytokines, cells were detached from the plate by incubating with PBS/EDTA, and viable cells were counted. Cell culture supernatants were collected to quantify, in an enzyme-linked immunoassay, the amount of VEGF and PlGF homodimers and VEGF/PlGF heterodimers secreted by different human melanoma cell lines (Table 1). Normal human keratinocytes, secreting both VEGF and PlGF polypeptides (B
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