Iron Chelators Inhibit the Growth and Induce the Apoptosis of Kaposi's Sarcoma Cells and of their Putative Endothelial Precursors
2000; Elsevier BV; Volume: 115; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2000.00119.x
ISSN1523-1747
AutoresThierry Simonart, Michel Heenen, Chantal Degraef, Graciela Andreï, Roger Mosselmans, Philippe Hermans, Jean‐Paul Van Vooren, Jean‐Christophe Noël, Johan R. Boelaert, Robert Snoeck,
Tópico(s)Viral-associated cancers and disorders
ResumoIron is suspected to be involved in the induction and/or progression of various human tumors. More particularly, iron may be involved in the pathogenesis of Kaposi's sarcoma, a tumor of probable vascular origin. This study was designed to investigate the effect of iron deprivation on Kaposi's sarcoma. The effects of iron chelators and iron deprivation associated with serum withdrawal were investigated on Kaposi's sarcoma-derived spindle cells, on a transformed Kaposi's sarcoma cell line (Kaposi's sarcoma Y-1) and on endothelial cells, which are the probable progenitors of Kaposi's sarcoma cells. Desferrioxamine and deferiprone, two chemically unrelated iron chelators, induced a time- and concentration-dependent inhibition of endothelial and Kaposi's sarcoma cell growth. The inhibition of cell growth was associated with a decrease in Ki-67 and in both stable and total proliferating cell nuclear antigen expression. Inhibition of the progression through the G1-phase of the cell cycle was further evidenced by decreased expression of cyclin D1 and of p34 cyclin-dependent kinase 4. Terminal deoxynucleotidyl transferase-mediated desoxyuridinetriphosphate nick end labeling assay, flow cytometry with annexin-V-fluorescein and morphologic analysis indicated that iron chelation also induced a time- and concentration-dependent apoptosis. This apoptotic effect was prevented by the addition of exogenous iron. Induction of iron deprivation in the culture medium by serum withdrawal led to similar cell cycle effects, which, however, could only be partly reverted by the addition of exogenous iron. In conclusion, these results show that iron deprivation inhibits the growth and induces the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors. This suggests that iron chelators may represent a potential therapeutic approach for the treatment of Kaposi's sarcoma. Iron is suspected to be involved in the induction and/or progression of various human tumors. More particularly, iron may be involved in the pathogenesis of Kaposi's sarcoma, a tumor of probable vascular origin. This study was designed to investigate the effect of iron deprivation on Kaposi's sarcoma. The effects of iron chelators and iron deprivation associated with serum withdrawal were investigated on Kaposi's sarcoma-derived spindle cells, on a transformed Kaposi's sarcoma cell line (Kaposi's sarcoma Y-1) and on endothelial cells, which are the probable progenitors of Kaposi's sarcoma cells. Desferrioxamine and deferiprone, two chemically unrelated iron chelators, induced a time- and concentration-dependent inhibition of endothelial and Kaposi's sarcoma cell growth. The inhibition of cell growth was associated with a decrease in Ki-67 and in both stable and total proliferating cell nuclear antigen expression. Inhibition of the progression through the G1-phase of the cell cycle was further evidenced by decreased expression of cyclin D1 and of p34 cyclin-dependent kinase 4. Terminal deoxynucleotidyl transferase-mediated desoxyuridinetriphosphate nick end labeling assay, flow cytometry with annexin-V-fluorescein and morphologic analysis indicated that iron chelation also induced a time- and concentration-dependent apoptosis. This apoptotic effect was prevented by the addition of exogenous iron. Induction of iron deprivation in the culture medium by serum withdrawal led to similar cell cycle effects, which, however, could only be partly reverted by the addition of exogenous iron. In conclusion, these results show that iron deprivation inhibits the growth and induces the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors. This suggests that iron chelators may represent a potential therapeutic approach for the treatment of Kaposi's sarcoma. Kaposi's sarcoma desferrioxamine p34 cyclin-dependent kinase 4 proliferating cell nuclear antigen human dermal microvascular endothelial cells Several data suggest that iron plays a part in the development of human cancers (Sussman, 1992Sussman H.H. Iron in cancer.Pathobiology. 1992; 60: 2-9Crossref PubMed Scopus (41) Google Scholar;Weinberg, 1996Weinberg E.D. The role of iron in cancer.Eur J Cancer Prev. 1996; 5: 19-36Crossref PubMed Scopus (2) Google Scholar). The scientific rationale to this came from the following: (i) iron is an essential element for dividing cells, because it is incorporated in numerous enzymes that play a part in DNA replication and cellular metabolism; (ii) tumor cells are relatively more iron deficient than normal cells, which is often reflected by an increased number of transferrin receptors on the cell surface (Page-Faulk et al., 1980Page-Faulk W. His B.L. Stevens P.J. Transferrin and transferrin receptors in carcinoma of the breast.Lancet. 1980; ii: 390-392Abstract Scopus (213) Google Scholar); (iii) iron may promote the formation of mutagenic hydroxyl radicals (Sussman, 1992Sussman H.H. Iron in cancer.Pathobiology. 1992; 60: 2-9Crossref PubMed Scopus (41) Google Scholar;Weinberg, 1996Weinberg E.D. The role of iron in cancer.Eur J Cancer Prev. 1996; 5: 19-36Crossref PubMed Scopus (2) Google Scholar); (iv) iron excess diminishes host defenses through inhibition of the activity of macrophages and lymphocytes (Weinberg, 1996Weinberg E.D. The role of iron in cancer.Eur J Cancer Prev. 1996; 5: 19-36Crossref PubMed Scopus (2) Google Scholar); (v) iron may induce the expression of anti-apoptotic signals in endothelial cells and hence, might trigger a ''switch'' to an angiogenic phenotype (Simonart et al. unpublished results). More particularly, we and colleagues have suggested that iron may act as a cofactor in the pathogenesis of Kaposi's sarcoma (KS), a tumor of probable vascular origin characterized by a prominent angiogenesis (Ziegler, 1993Ziegler J.L. Endemic KS in Africa and local volcanic soils.Lancet. 1993; 342: 1348-1351Abstract PubMed Scopus (127) Google Scholar;Simonart et al., 1998Simonart T. Noel J.C. Andrei G. et al.Iron as a potential cofactor in the pathogenesis of Kaposi's sarcoma.Int J Cancer. 1998; 78: 720-726Crossref PubMed Scopus (43) Google Scholar). This hypothesis may partly explain the high prevalence of KS in geographic areas with iron oxide-rich volcanic clays, such as Sicily, Iceland, and the East African Rift system (Ziegler, 1993Ziegler J.L. Endemic KS in Africa and local volcanic soils.Lancet. 1993; 342: 1348-1351Abstract PubMed Scopus (127) Google Scholar;Hjalgrim et al., 1998Hjalgrim H. Tulinius H. Dalberg J. Hardason S. Frisch M. Melbye M. High incidence of classical KS in Iceland and the Faroe Islands.Br J Cancer. 1998; 77: 1190-1193Crossref PubMed Scopus (25) Google Scholar;Simonart et al., 1998Simonart T. Noel J.C. Andrei G. et al.Iron as a potential cofactor in the pathogenesis of Kaposi's sarcoma.Int J Cancer. 1998; 78: 720-726Crossref PubMed Scopus (43) Google Scholar,Simonart et al., 1999Simonart T. Van Vooren J.P. Herbauts J. Boelaert J.R. High prevalence of KS in Iceland and the Faroe Islands.Br J Cancer. 1999; 79: 373PubMed Google Scholar). It may also provide a nonhormonal explanation for the lower prevalence of KS among women (Lunardi-Iskandar et al., 1995Lunardi-Iskandar Y. Bryant J.L. Zeman R.A. et al.Tumorigenesis and metastasis of neoplastic KS cell line in immunodeficient mice blocked by a human pregnancy hormone.Nature. 1995; 375: 64-68Crossref PubMed Scopus (213) Google Scholar), as they are known to have lower iron reserves than men (Kushner et al., 1988Kushner J.P. Hypochromic anemia.in: Wyngaarden J.B. Smith L.H. Cecil, Textbook of Medicine. 18th edn. W.B. Saunders, Philadelphia1988: 892-900Google Scholar). Based on the probable role of iron in tumor development, the iron chelator desferrioxamine (DFO) is currently investigated in the management of human tumors. Promising results were obtained with various tumor cell types (Estrove et al., 1987Estrove B. Tawa A. Wang O.H. et al.In vitro and in vivo effects of deferoxamine in neonatal acute leukemia.Blood. 1987; 69: 757-761PubMed Google Scholar;Donfrancesco et al., 1990Donfrancesco A. Deb G. Dominici C. Pileggi D. Castello M.A. Helson L. Effects of single course of deferoxamine in neuroblastoma patients.Cancer Res. 1990; 50: 4929-4930PubMed Google Scholar;Weinberg, 1996Weinberg E.D. The role of iron in cancer.Eur J Cancer Prev. 1996; 5: 19-36Crossref PubMed Scopus (2) Google Scholar). Depending on the studied cell models, DFO has been shown to inhibit replication, induce apoptosis, and/or differentiation. DFO arrests cell cycle by a mechanism that is not completely understood and that may be related to the inhibition of the ribonucleotide reductase, which possesses a labile iron prosthetic group (Lederman et al., 1984Lederman H.M. Cohen A. Lee J.W. Freedman M.H. Gelfand E.W. Desferrioxamine: a reversible S-phase inhibitor of human lymphocyte proliferation.Blood. 1984; 64: 748-753Crossref PubMed Google Scholar;Barankiewicz and Cohen, 1987Barankiewicz J. Cohen A. Impairment of nucleotide metabolism by iron-chelating deferoxamine.Biochem Pharmacol. 1987; 36: 2343-2347Crossref PubMed Scopus (18) Google Scholar). Whereas there is no doubt that the requirement for iron is due in part to the needs of this enzyme, it does not explain why iron chelators such as DFO block cells in G1, before nucleotide reductase is required. DFO also inhibits DNA repair processes (Kaplinsky et al., 1987Kaplinsky C. Estrov Z. Freedman M.H. Gelfand E.W. Cohen A. Effect of deferoxamine on DNA synthesis, DNA repair, cell proliferation, and differentiation of HL-60 cells.Leukemia. 1987; 1: 437-441PubMed Google Scholar). The effect of DFO on DNA synthesis and repair may be reflected in studies that have shown treatment with DFO induces apoptosis in rapidly growing tumor cells (Fukuchi et al., 1994Fukuchi K. Tomoyasu S. Tsuruoka N. Gomi K. Iron deprivation-induced apoptosis in HL-60 cells.FEBS Lett. 1994; 350: 139-142Abstract Full Text PDF PubMed Scopus (86) Google Scholar;Haq et al., 1995Haq R.U. Werely J.P. Chitambar C.R. Induction of apoptosis by iron deprivation in human leukemic CCRF-CEM cells.Exp Hematol. 1995; 23: 428-432PubMed Google Scholar;Hileti et al., 1995Hileti D. Panayiotidis P. Hoffbrand A.V. Iron chelators induces apoptosis in proliferating cells.Br J Haematol. 1995; 89: 181-187Crossref PubMed Scopus (114) Google Scholar). In contrast, DFO does not cause death of nondividing lymphocytes or granulocytes, implying its relative specificity towards proliferating cells (Hileti et al., 1995Hileti D. Panayiotidis P. Hoffbrand A.V. Iron chelators induces apoptosis in proliferating cells.Br J Haematol. 1995; 89: 181-187Crossref PubMed Scopus (114) Google Scholar). In this study, we investigated the effects of iron deprivation on KS-derived spindle cells, on an immortalized KS cell culture (KS-Y1) and on human dermal microvascular endothelial cells (HDMEC), which are the putative progenitors of cutaneous KS. Iron chloride (FeCl3) was obtained from FlU.K.a (Buchs, Switzerland). Ferric nitriloacetate, dimethylsulfoxide, and N-acetylcysteine were from Sigma (Bornem, Belgium). DFO was purchased as its commercially available mesylate salt. 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone, L1) was kindly provided by Dr. Stembert (Duchefa Farma, Haarlem, the Netherlands). Bax monoclonal antibody (MoAb) (clone G206–1276) was from Pharmingen (San Diego, CA); Bcl-2 MoAb (clone 100) and p34 cyclin-dependent kinase 4 (p34cdk4) MoAb (DCS32.2 clone) from Oncogene Research Products (Cambridge, MA); cyclin D1 MoAb (P2D11F11 clone) from Novocastra (Newcastle, U.K.); and proliferating cell nuclear antigen (PCNA) MoAb (PC10 clone) from Boehringer Mannheim (Mannheim, Germany). Normal immunoglobin fractions were from Dako (Glostrup, Denmark). KS cell cultures were derived from cutaneous nodular KS lesions from an acquired immune deficiency syndrome patient (KS-1), a renal transplant recipient (KS-2), and a patient with sporadic KS (KS-3). These three cultures were established similarly as previously described (Benelli et al., 1994Benelli R. Repetto L. Carlone S. Parravicini C. Albini A. Establishment and characterization of two new Kaposi's sacoma cell cultures from an AIDS and a non-AIDS patient.Res Virol. 1994; 145: 251-259Crossref PubMed Scopus (31) Google Scholar). Briefly, fresh lesions were minced in small pieces and enzymatically digested with collagenase (1 mg per ml) from Clostridium histolyticum for 90 min. Cells were grown without additional growth factors in minimum essential medium D-Val (Gibco, Paisley, Scotland) containing 10% inactivated fetal bovine serum (FBS) (Gibco), 1% glutamine, 1% nonessential amino acids, penicillin (100 U per ml), and streptomycin (100 μg per ml). Cells isolated from these cultures were characterized as KS cells morphologically (spindle-shaped morphology) and immunohisto- chemically (positivity for laminin, vimentin, collagen IV, α-smooth muscle actin, ICAM-1 and Bcl-2 protein, and negativity for cytokeratin, von Willebrand factor, ICAM-2, ELAM-1, VCAM-1, CD4, CD34, CD40, CD45), in accordance with previous published reports (Benelli et al., 1994Benelli R. Repetto L. Carlone S. Parravicini C. Albini A. Establishment and characterization of two new Kaposi's sacoma cell cultures from an AIDS and a non-AIDS patient.Res Virol. 1994; 145: 251-259Crossref PubMed Scopus (31) Google Scholar;Pammer et al., 1996Pammer J. Plettenberg A. Weninger W. et al.CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma.Am J Pathol. 1996; 148: 1387-1396PubMed Google Scholar;Simonart et al., 1998Simonart T. Degraef C. Noel J.C. et al.Overexpression of Bcl-2 in Kaposi's sarcoma-derived cells.J Invest Dermatol. 1998; 111: 349-353Crossref PubMed Scopus (26) Google Scholar). In addition, we have shown that these cells constitutively produce a 92 kDa type IV collagenase (Blankaert et al., 1998Blankaert D. Simonart T. Van Vooren J.P. et al.Constitutive release of metalloproteinase-9 (92-kDa type IV collagenase) by Kaposi's sarcoma cells.J Acquired Immune Defic Syndr. 1998; 18: 203-209Crossref Scopus (35) Google Scholar), which suggests they have an invasive phenotype and that they may adequately represent the tumor cells of KS. The experiments presented here were performed on cell cultures between the fifth and the fifteenth passage. The immortalized KS-Y1 cell line was kindly provided by Dr Y. Lunardi-Iskandar (Institute of Human Virology, Baltimore, MD). It was maintained in RPMI (Gibco) containing 10% inactivated FBS (Gibco), 1% glutamine, 1% nonessential amino acids, 1% sodium pyruvate, penicillin (100 U per ml) and streptomycin (100 μg per ml). No exogenous growth factor was added. HDMEC were cultured by a method similar to the one reported byKluger et al., 1997Kluger M.S. Johnson D.R. Pober J.S. Mechanism of sustained E-selectin expression in human dermal microvascular endothelial cells.J Immunol. 1997; 158: 887-896PubMed Google Scholar. HDMEC were isolated from normal adult breast skin obtained as discarded tissue from reduction mammoplasties (Department of Plastic Surgery, Erasme University Hospital, Brussels). Informed consent was obtained from the patients. Fresh skin was stretched flat and sectioned horizontally. After an 80 min incubation in 2 mg dispase II per ml (Boehringer) at 37°C, the epidermis was peeled off and cells from both sides of the underlying epidermis were gently scraped into RPMI (Gibco) and filtered through a 70 μM nylon mesh. The filtrate, containing single cells, was washed once in minimum essential medium D-Val and plated on to tissue culture plastic precoated with the cell culture medium (C-22020, PromoCell, Heidelberg, Germany) supplemented with 10% FBS (Gibco). HDMEC were allowed to attach approximately 4 h before gentle aspiration (to remove cell debris) and addition of fresh media. The cells were used at passages 5–12. Fluorescence-activated cell sorter analysis revealed that > 95% of the cell population was positive for endoglin. Immunostaining with a specific anti-human fibroblast antibody (Dianova, Hamburg, Germany) (Romero et al., 1997Romero L.I. Zhang D.N. Herron G.S. Karasek M.A. Interleukin-1 induces major phenotypic changes in human skin microvascular endothelial cells.J Cell Physiol. 1997; 173: 84-92Crossref PubMed Scopus (64) Google Scholar) was negative. Briefly, exponentially growing cells were allowed to proliferate in complete growth medium until they reached 85–90% confluence. Iron deprivation was induced in subconfluent cell cultures by serum deprivation. After having removed the FBS-containing medium, serum-free RPMI (with HEPES and glutamine) (Biowhittaker, Verviers, Belgium) supplemented with 1% nonessential amino acids was placed on to the monolayer cultures. Iron chelators (DFO and deferiprone, concentrations as indicated) were added either to subconfluent cell cultures containing 10% FBS or to serum-deprived cell cultures. The effect of the drugs on cellular proliferation was assayed as previously described (Ferguson and Cheng, 1989Ferguson P.J. Cheng Y. Phenotypic instability of drug sensitivity in a human colon carcinoma cell line.Cancer Res. 1989; 49: 1148-1153PubMed Google Scholar;Andrei et al., 1991Andrei G. Snoeck R. Schols D. Goubau P. Desmyter J. De Clercq E. Comparative activity of selected antiviral compounds against clinical isolates of human cytomegalovirus.Eur J Clin Microbiol Infect Dis. 1991; 10: 1026-1033Crossref PubMed Scopus (68) Google Scholar). Briefly, cells were seeded at 3 × 103 cells per well in a volume of 0.1 ml into 96-well microtiter plates. The cells were incubated for 1 d to allow entry into the exponential growth phase and were then pulsed with various concentrations of the tested compounds. After 7 d incubation at 37°C in 5% CO2, the cell number was determined with a Coulter counter. The IC50 (defined as the concentration required to reduce cell growth by 50%) was determined by interpolation of plotted data. Viable cell numbers were estimated by the Trypan blue exclusion test. At various time points, cells in individual monolayer cultures were harvested and made into single-cell suspensions by trypsinization. The cell suspensions were then incubated for 3 min with Trypan blue dye. The cells remaining negative to the blue dye staining after Trypan blue exclusion were counted as viable cells. The ability of the cells to survive and further divide was determined by the retention of their colony-forming ability upon return to serum-supplemented conditions. Cells remaining adherent to the plate were harvested at various time intervals after the serum withdrawal and equal volumes from control and DFO-exposed cultures were resuspended in complete growth medium containing 10% FBS. Plating efficiency was determined at various time intervals as the total number of cells per dish and as the number of colonies ≥ 4 cells per 10 high power fields. Immunohistochemistry was carried out on cytospins. Briefly, the cells were harvested at various time points and made into a single-cell suspension by trypsinization. For Bax (I-19), the cytospin preparations were fixed in Bouin for 15 min and then incubated in 3% (vol/vol) H2O2 in phosphate-buffered saline (PBS) for 30 min at room temperature. The samples were incubated in normal goat serum for 30 min to block nonspecific interactions, and then anti-Bax antibody was applied at the dilution of 2 μg per ml overnight at 4°C. Normal rabbit immunoglobin fractions at the same concentration as the primary antibodies served as negative controls. Immunohistochemical staining was achieved with the PK-4001 Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxylin. For Bcl-2 MoAb, two different fixation procedures were used. (i) The cells were fixed in formalin for 1 h at room temperature and incubated in 3% (vol/vol) H2O2 in PBS for 30 min. They were then microwaved at 750 W for 2 × 5 min in a citrate buffer, pH 6 and then blocked with normal goat serum 1/20 for 30 min. (ii) They were fixed in absolute methanol for 10 min at room temperature, incubated in 3% (vol/vol) H2O2 in PBS for 30 min, and then blocked with normal goat serum. The cytospins were then immersed for 10 min in PBS before immunocytochemical staining with 5 μg per ml Bcl-2. For stable PCNA, the samples were fixed in absolute methanol for 10 min and permeabilized with Triton-X100 (0.1% in PBS), rinsed twice with PBS and incubated in 3% (vol/vol) H2O2 in PBS for 30 min before immunocytochemical staining (PC10, 5 μg per ml). For total (i.e., stable and labile) PCNA they were fixed in 1% paraformaldehyde in PBS (pH 7.2), containing 6.6 μg of lysolecithin, followed by immersion for 10 min in absolute methanol at ice temperature and permeabilization with 0.1% Nonidet P-40 in PBS on ice, as previously described (Galand et al., 1995Galand P. Del Bino G. Morret M. Capel P. Degraef C. Fokan D. Feremans W. PCNA immunopositivity index as a substitute to 3H-thymidine pulse-labeling index (TLI) in methanol-fixed human lymphocytes.Leukemia. 1995; 9: 1075-1084PubMed Google Scholar). For cyclin D1, the preparations were fixed with acetone/ethanol (1:1) for 10 min at room temperature. The formed immune complexes were detected with Envision/HRP (Dako). Peroxidase activity was developed with 3,3′-diaminobenzidine tetrahydrochloride (0.012% in PBS) and H2O2 (0.1%) and the cells were counterstained with hematoxylin. Confluent cell cultures were washed with complete PBS and lyzed in sodium dodecyl sulfate buffer (5% β-mercaptoethanol, 10% glycerol, 80 mM sodium dodecyl sulfate, 60 mM Tris, pH 6.8). For each sample, a total quantity of 40 μg of protein was electrophoresed on a 12% sodium dodecyl sulfate–polyacrylamide linear gradient slab gel, in Tris-buffered saline (TBS) solution. The proteins were then electrophoretically transferred to nitrocellulose sheets. The blots were subsequently incubated for 1 h in 1% fat-free milk in TBS and for 18 h either with Bcl-2 MoAb at a 2-μg per ml concentration in TBS (20 mM Tris, 125 mM NaCl) or with Bax MoAb at a 4 μg per ml concentration or with p34cdk4 MoAb at a 2.5 μg per ml concentration. After extensive washing in TBS, the immune complexes were detected with either a biotinylated antirat immunoglobulin (Bax) or an anti-mouse immuno- globulin (Bcl-2 and p34cdk4) (diluted 1:250 in TBS) (Amersham), serving as binding bridge to biotin–streptavidin peroxidase preformed complexes used at the same dilution. Preformed complexes were detected by photographic recording of the chemiluminescence (ECL) emitted by a H2O2-reacting probe, using the BM Chemiluminescence Blotting Kit (Boehringer Mannheim). Lysates from KS-derived cells and from MCF-7 cells were used as positive controls for Bcl-2 (Simonart et al., 1998Simonart T. Noel J.C. Andrei G. et al.Iron as a potential cofactor in the pathogenesis of Kaposi's sarcoma.Int J Cancer. 1998; 78: 720-726Crossref PubMed Scopus (43) Google Scholar). Lysates from NIH-3T3 fibroblasts served as a positive control for Bax (Miyashita et al., 1995Miyashita T. Kitada S. Krajeweski S. Horne W.A. Delia D. Reed J.C. Overexpression of the Bcl-2 protein increases the half-life of p21Bax.J Biol Chem. 1995; 270: 26049-26052Crossref PubMed Scopus (99) Google Scholar). The immunoblots were scanned with a Microtek Phantom 4800 apparatus. TdT assay for strand breaks was performed similarly as previously reported (Heenen et al., 1998Heenen M. Laporte M. Noel J.C. Degraef C. Methotrexate induces apoptotic cell death in human keratinocytes.Arch Dermatol Res. 1998; 290: 240-245Crossref PubMed Scopus (60) Google Scholar). Briefly, formalin-fixed cytospin preparations were incubated with 20 μg per ml proteinase K (Sigma) for 15 min at room temperature in order to strip the nuclei from proteins. The samples were then washed four times in double-distilled water for 2 min. Endogenous peroxidase was inactivated by covering the slides with 2% (vol/vol) H2O2 in PBS for 30 min at room temperature. The preparations were rinsed with double-distilled water, and immersed in TdT labeling buffer (30 mM Trisma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). TdT (0.3 e.u. per μl), dATP, and biotinylated dUTP in TdT buffer were then added to cover the cytospin preparations in a humid atmosphere at 37°C for 60 min. The reaction was terminated by transferring the slides to terminating buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min at room temperature. The slides were then rinsed with double-distilled water, covered with a 2% aqueous solution of bovine serum albumin for 10 min at room temperature, rinsed in double-distilled water and immersed in PBS for 5 min. Peroxidase activity was developed with 3,3′-diaminobenzidine and H2O2. The analysis of phosphat- idylserine on the leaflet of apoptotic cell membranes was performed by using annexin-V fluorescein (Boehringer Mannheim) and propidium iodide for the differentiation from necrotic cells. Briefly, cells were seeded at 5 × 104 cells per well in a volume of 2 ml into six-well plates. The cells were incubated for 1 d to allow entry into the exponential growth phase and were then pulsed with various concentrations of DFO. After 2 and 5 d incubation at 37°C in 5% CO2, the cells were harvested with ethylenediamine tetraacetic acid/PBS. After rinsing the cells with PBS, the cell pellets were resuspended in 100 μl of the staining solution (20 μl annexin-V-fluorescein labeling reagent in 1 ml HEPES buffer (HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) and 20 μl propidium iodide (50 μg per ml). After a 15 min incubation period, the cells were analyzed on a FACStar Plus (Becton Dickinson, San Jose, CA). The Student's t test (two-tailed) was used to compare different groups of data that were obtained from three sets of experiments. Doubling times of the different cell cultures during exponential growth phase are shown in Table 1A. All three examined KS-derived spindle cell cultures exhibited similar growth properties, which is in agreement with previous studies showing that the spindle cell component of the different epidemiologic settings of KS shares common biologic and/or pathologic characteristics (Benelli et al., 1994Benelli R. Repetto L. Carlone S. Parravicini C. Albini A. Establishment and characterization of two new Kaposi's sacoma cell cultures from an AIDS and a non-AIDS patient.Res Virol. 1994; 145: 251-259Crossref PubMed Scopus (31) Google Scholar;Kaaya et al., 1995Kaaya E.E. Parravicini C. Ordonez C. Gendelman R. Berti E. Gallo R.C. Biberfeld P. Heterogeneity of spindle cells in KS. comparison of cells in lesions and in culture.J Acquired Immune Defic Syndr. 1995; 10: 295-305Google Scholar). To analyze further the differential kinetics of the cells, we studied their expression of the proliferation markers, PCNA and Ki-67 antigen. There was a positive correlation between the basal growth rates and the Ki-67 antigen and total (formalin-fixed) PCNA indexes of the different cell types (Table 2A), suggesting that the long doubling times of the KS-derived cell population may be related to a high proportion of cells in the G0 or G0/G1 phase of the cell cycle rather than to increased cell cycle traverse periods.Table IKinetic parameters of untreated and bFGF-treated KS-derived cells, KS-Y1 cells, and HDMECKS-1KS-2KS-3KS-Y1HDMEC(A) Doubling times (h) and proliferation indexes (% of positive cells) of KS-derived cells, KS-Y1 cells, and HDMECaMean ± SEM (n = 3).Doubling times85 ± 2095 ± 2691 ± 2529 ± 548 ± 10Total PCNA index53 ± 2133 ± 1037 ± 1473 ± 858 ± 8Ki-67 index49 ± 2025 ± 1235 ± 870 ± 961 ± 10(B) Doubling times (h) of KS-derived cells, KS-Y1 cells, and HDMEC cultured with bFGF (0.002 μg per ml)aDoubling times46 ± 1060 ± 1853 ± 1830 ± 545 ± 12a Mean ± SEM (n = 3). Open table in a new tab Table IIIC50 (μM) valuesaMean ± SEM (n = 3). of DFO for KS-derived cells, KS-Y1 cells, and HDMEC, determined 3 and 7 d after the addition of the drugConditionKS-1KS-2KS-3KS-Y1HDMECControlday 357 ± 17115 ± 5585 ± 297 ± 225 ± 7+bFGF (0.002 μg per ml)21 ± 8bValues shown with + bFGF are calculated relative to the corresponding control cells treated with bFGF.*p < 0.05 as compared with control cells.45 ± 2231 ± 10*7 ± 322 ± 6Controlday 726 ± 840 ± 1434 ± 94 ± 212 ± 4+bFGF (0.002 μg per ml)10 ± 3*17 ± 712 ± 4*5 ± 39 ± 3a Mean ± SEM (n = 3).b Values shown with + bFGF are calculated relative to the corresponding control cells treated with bFGF.* p < 0.05 as compared with control cells. Open table in a new tab The effects of the iron chelators DFO and deferiprone were first studied on exponentially growing cells. DFO induced a concentration-dependent inhibition of endothelial and KS cell growth. Analysis of the IC50 values (defined as the concentration of the drug required to reduce cell growth by 50% and determined by interpolation of plotted data) indicated that there was a positive correlation (p < 0.01 using Spearman's correlation test) between the basal cell growth of the different cell types and the inhibitory effect of DFO (Table 2), suggesting that DFO may act through a nonspecific effect on cell division. The IC50 values exhibited a significant decrease between days 3 and 7, indicating a time-dependent inhibitory effect of DFO (Table 2). Similar effects were obtained with equivalent iron-binding concentrations of deferiprone (data not shown). No significant inhibition of cell growth could be observed when the iron chelators were incubated with stochiometric amounts of FeCl3. To test the possibility that the differences in IC50 values were related to different doubling times, we investigated the effect of iron chelation under stimulation with basic fibroblast growth factor (bFGF), which is one of the major autocrine growth factors for KS cells (Ensoli et al., 1994Ensoli B
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