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Reversal of malignant phenotype in human osteosarcoma cells transduced with the alkaline phosphatase gene
Bone Vol. 26, No. 3 March 2000:215–220
ORIGINAL ARTICLES
Reversal of Malignant Phenotype in Human Osteosarcoma Cells Transduced With the Alkaline Phosphatase Gene M. C. MANARA,1 N. BALDINI,1 M. SERRA,1 P.-L. LOLLINI,2 C. De GIOVANNI,2 M. VACCARI,3 A. ARGNANI,3 S. BENINI,1 D. MAURICI,1 P. PICCI,1 and K. SCOTLANDI1 1
Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy Istituto di Cancerologia, Universita` degli Studi di Bologna, Bologna, Italy 3 IST, Istituto di Ricerche per la Ricerca sul Cancro-Genova, Unita` Satellite di Biotecnologie, Bologna, Italy 2
in fundamental biological processes. However, as yet, the physiological role of L/B/K ALP is unknown, except that the bone isoenzyme is thought to play an essential role in normal skeletal mineralization. Bone ALP is anchored to the outer plasma membrane of osteoblasts by a proteoglycan-inositol linkage to phosphatidylinositol8 and can be released from the cell surface by a glycan-inositol hydrolase and/or surrounded by membrane vesicles.5,13 Evidence for a role of bone ALP in mineralization derives from the fact that the introduction of bone ALP cDNA into ALP-negative cells confers an in vitro capacity for mineralization,28 and that missense mutations of bone ALP gene are associated with a lethal disease characterized by osteomalacia and a deficiency of L/B/K ALP in all tissues (placental and intestinal ALP levels being unaffected).26 However, the precise biochemical function of bone ALP activity is still unknown, and its role in normal bone physiology is not completely understood. Even less defined is its clinical significance in osteosarcoma, a bone-forming neoplasm showing a wide range of expression of ALP. With regard to osteosarcoma, we have previously demonstrated the existence of an inverse correlation between cellular bone ALP expression and tumor aggressiveness.21 In this paper, we further investigated this relationship by transfecting the L/B/K ALP gene25 in an osteosarcoma cell line with no evidence of basal expression of this enzyme.23 The in vitro and in vivo behavior of selected transfectants were analyzed with regard to their invasive, metastatic, and tumorigenic ability.
Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the boneforming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme. (Bone 26:215–220; 2000) © 2000 by Elsevier Science Inc. All rights reserved. Key Words: Alkaline phosphatase; Osteosarcoma; Matrix metalloproteinase; Tumorigenicity; Metastasis; Athymic mice. Introduction
Material and Methods
Alkaline phosphatases (ALP) (orthophosphoric-monoester phosphohydrolase) are a group of cell surface glycoproteins that hydrolize a broad range of monophosphate esters at an optimum alkaline pH. Based on heat stability, sensitivity to inhibitors, antigenicity, and electrophoretic mobility, four major ALP isoenzymes have been characterized so far,8 including placental, intestinal, liver/bone/kidney (L/B/K), and germ cell ALP, each encoded by separate genes. Each form of human ALP exhibits a characteristic pattern of tissue distribution, placental and intestinal ALP being found predominantly in the tissues for which they are named, whereas L/B/K ALP is widely distributed. Alkaline phosphatases are found in organisms ranging from bacteria to mammals, and their conserved presence suggests an involvement
Cell Lines U-2 OS human osteosarcoma cells were routinely cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with penicillin (100 U/mL), streptomycin (100 g/mL) (Life Technologies, Paisley, Scotland), and 10% heat-inactivated fetal calf serum (FCS; Biological Industries, Kibbutz Beth Haemek, Israel). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. The transfection of U-2 OS cell line was performed by calcium phosphate coprecipitation 24 h after seeding of 105 cells in a 100-mm dish.27 Cells were cotransfected with 10 g of pSV2Aalp⬘, an expression vector containing the full-lenght L/B/K ALP cDNA26 and 10 g of pSV2Neo, containing the neo gene, which confers resistance to neomycin. After 24 h of DNA exposure, medium was removed and cells were maintained in normal growth medium for 2 more days. Cells were then subjected to selective medium containing 500 g/mL of G418
Address for correspondence and reprints: Dr.ssa K. Scotlandi, Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via Di Barbiano 1/10, 40126 Bologna, Italy. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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(Sigma, St. Louis, MO) and cultured for 2 weeks with a selective medium change every 2 days. Individual colonies were cloned with glass cylinders and expanded in selective medium. U-2/ALP3, U-2/ALP23, U-2/ALP28, and U-2/ALP40 are representative of ALP cotransfected clones and were chosen for their different range of ALP activity. To obtain additional controls, U-2 OS cells were transfected with pSV2Neo alone and selected in medium containing G418 (500 g/mL). U-2/Neo8 and U-2/Neo26 are representative of this series of transfectants.
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S.A., Marseilles, France), 1:10 dilution; GC-4 (anti-A-CAM; Sigma), 1:100 dilution; CDw49b VLA2 (anti-alpha 2 chain, ␣21; Immunotech S.A.), 1:10 dilution; P1B5 (anti-alpha3 chain, ␣31; Oncogene Research Product) 1:20 dilution; CDw49d VLA4 (anti-alpha 4 chain, ␣41; Immunotech S.A.), 1:10 dilution; CDw49e VLA5 (anti-alpha 5 chain, ␣51; Immunotech S.A.), 1:10 dilution; and CDw49f VLA6 (anti-alpha 6 chain, ␣61; Immunotech S.A.), 1:10 dilution. Chemotaxis Assay
In Vitro Growth Features For the determination of population doubling time, 0.5 ⫻ 106 cells were seeded in IMDM plus 10% FCS in 25-cm2 flasks (Falcon, Oxnard, CA) and harvested daily by using 0.25% trypsin/0.002% EDTA (Life Technologies, Paisley, Scotland). Doubling time was calculated during the logarithmic growth phase. Cell viability was determined by Trypan blue dye exclusion. For the evaluation of cloning efficiency, 200 –1600 cells were seeded in 60-mm dishes. After 7 days, cells were fixed with methanol for 10 min at room temperature, and stained with Giemsa. Colonies of at least 10 cells were counted using an inverted microscope. Soft-Agar Assay Anchorage-independent growth was determined in 0.33% agarose (FMC Bio Products, Rockland, ME) with a 0.5% agarose underlay. Cell suspensions (10,000 –33,000 cells per 60-mm dish) were plated in a semi-solid medium (IMDM with 10% FCS containing 0.33% agarose). Colonies were counted after 14 days. ALP Activity The percentage of cells displaying ALP activity at their cell surface was evaluated on cytospins obtained 4 days after cell seeding using a cytochemical method (Kit 86R; Sigma, St. Louis, MO). The percentage of positive cells was calculated on at least 300 cells. Cellular ALP activity was also analyzed 4 days after seeding. Approximately 106 cells were resuspended in homogenization buffer (1 mmol/L MgCl2, 1 mmol/L CaCl2, 20 mol/L ZnCl2, 0.1 mol/L NaCl, 0.1% Triton X-100, 0.05 mol/L TrisHCl, pH 7.4) and disrupted by pipetting. The homogenate was used for the ALP assay, which was performed with p-nitrophenol phosphate as a substrate, according to the instructions of the manufacturer (Sigma). L/B/K ALP activity was normalized for the content of protein in the sample. Proteins were measured according to the Bradford method2 using bovine serum albumin (BSA) fraction V (Sigma) as a standard. In particular, one unit of L/B/K ALP activity is defined as the amount of enzyme capable of transforming 1 mol of substrate in 1 min at 25°C. L/B/K ALP activity in the conditioned medium due to the released ALP was also measured 4 days after cell seeding using p-nytrophenyl phosphate as a substrate, in accordance to the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany). Phenotypic Characteristics The expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-3 (LFA-3), and A-cell adhesion molecule (A-CAM) as well as of ␣21, ␣31, ␣41, ␣51, and ␣61 integrins was determined by flow cytometry after indirect immunofluorescence using the following monoclonal antibodies: LFA-3 (anti-LFA-3; Immunotech S.A., Marseille, France), 1:20 dilution; CD54 ICAM (anti-ICAM-1; Immunotech
The chemotaxis assay was made using Transwell chambers (Costar, Cambridge, MA) as previously described.4 Briefly, 1.5 ⫻ 105 cells were resuspended in Dulbecco’s modified medium (Life Technologies, St. Louis, MO) containing 0.1% BSA (Sigma) and seeded in the upper compartment of the chamber. A 24-h supernatant from BALB/c 3T3 cells collected in the absence of serum was placed in the lower compartment as a source of chemoattractant. The two compartments were separated by an 8.0-m pore size, polyvinylpirrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA) coated with 5 g/mL gelatin (Sigma). Cells were allowed to migrate for 6 h at 37°C. After incubation, cells that had migrated to the lower side of the filter were fixed in ethanol and stained with toluidine blue. Five to 10 fields/filter were counted at ⫻160. Three different experiments were made for each cell line. Chemoinvasion Assay This assay is a modification of the chemotaxis assay.1 Polycarbonate filters coated with Matrigel (16.5 g/filter; Collaborative Biomedical Products, Bedford, MA) were placed in chemotaxis chambers. After 6 h of incubation, cells on the lower surface of filter were scored as for chemotaxis. Three different experiments were made for each cell line. Detection of Matrix Metalloproteinase-2 and -9 Activity Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated on confluent monolayers of U-2 OS cells and ALP transfectants by replacing the culture medium with fresh serumfree medium. Supernatants were collected 24 h later and, to evaluate MMP-9 activity, were concentrated with a Centricon Plus-20 centrifugal filter devise provided with an ultrafiltration membrane with a 30,000 nominal molecular weight cut-off limit (Millipore Corporation, Bedford, MA). Matrix metalloproteinase-2 and 9 activity was determined by the enzyme-linked immunosorbent assay (ANOVA) test Biotrak MMP-2 or MMP-9 activity assay systems (Amersham Pharmacia Biotech, Milan, Italy). The test was performed according to the manufacturer’s instructions. Briefly, standards and samples were incubated at 4°C overnight in microtiter wells precoated with anti-MMP-2 or anti-MMP-9 antibody. After extensive washings, any bound MMP-2 or MMP-9 was activated by adding aminophenylmercuric acetate. Active MMP-2 or MMP-9 was then detected through the activation of a modified urokinase proenzyme and the subsequent cleavage of its chromogenic peptide substrate. The resultant color was read at 405 nm, and the concentration of active MMP-2 or MMP-9 was determined by interpolation from the standard curve. In Vivo Growth Female 4 –5-week-old Cr1:nu/nu (CD-1)BR athymic mice (Charles River Italia, Como, Italy) were used. Tumorigenicity was determined after subcutaneous injection of 30 ⫻ 106 cells.
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M. C. Manara et al. Alkaline phosphatase and malignancy in osteosarcoma cells
Table 1. In vitro ALP activity of U-2 OS cell line and ALP-transfected clones measured 4 days after cell seeding
Cell line
Expression of membrane-bound ALP (% of positive cells/total)
Membrane-bound ALP activity (mU/mg protein)
ALP activity in conditioned medium (mU/ mL/106 cells)
0 0 0 15 ⫾ 7 100 100 100 100
0 0 0 14.5 ⫾ 0.5 46.0 ⫾ 5.0 105.5 ⫾ 5.5 81.5 ⫾ 12.5 104.7 ⫾ 18.4
0.2 0.3 0.2 4.4 ⫾ 1.6 4.2 ⫾ 0.8 102.7 ⫾ 18.7 136.5 ⫾ 38.7 120.8 ⫾ 20.8
U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40 Saos-2
Tumor growth was assessed once weekly by measuring tumor volume, calculated as /6 ⫻ [公(ab)]3, where a and b are the two major diameters.12 Mice were killed and autopsied when mean tumor volume was 5 cc or, in mice with no evidence of tumor, at 6 months after cell inoculation. Histological sections obtained from the tumors growing in athymic mice were stained with hematoxylin-eosin. Experimental metastatic ability was evaluated after injection of 2 ⫻ 106 cells in a tail lateral vein. To obtain natural killer-depressed animals, mice were injected intravenously with 0.4 mL of a 1:25 dilution of anti-asialo GM1 antiserum (Wako, Du¨sseldorf, Germany) 24 h before cell inoculation. Two months later, mice were killed and the number of pulmonary metastases was determined by counting with a stereomicroscope after staining with black India ink. Statistical Analysis Student’s t test and ANOVA were used for repeated measurement values. For frequency data, Fisher’s, Mann-Whitney, and ANOVA rank sum tests were used.
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Table 2. In vitro growth parameters of U-2 OS cell line and ALPtransfected clones
Cell line
Doubling time (h)
Cloning efficiencya (%)
No. of colonies in soft agarb
18.3 ⫾ 0.2 19.2 ⫾ 1.2 21.9 ⫾ 3.6 24.1 ⫾ 6.7 23.5 ⫾ 4.1 27.1 ⫾ 4.6 26.1 ⫾ 5.8
59.7 ⫾ 1.7 55.7 ⫾ 5.8 52.5 ⫾ 3.1 51.9 ⫾ 2.8 50.1 ⫾ 4.2 58.4 ⫾ 1.9 50.6 ⫾ 0.4c
648 ⫾ 55 540 ⫾ 20 657 ⫾ 55 495 ⫾ 20 324 ⫾ 7d 391 ⫾ 7d 316 ⫾ 51d
U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40 a
Cloning efficiency was determined by counting the colonies 1 week after seeding with 200 –1,600 cells/cm2. Data are expressed as mean of triplicate plates ⫾ SE. b 10,000 cells were plated in 0.33% agarose with IMDM plus 10% FCS. Data are expressed as mean of triplicate plates ⫾ SE. c p ⬍ 0.05, with respect to U-2 OS cell line, by paired t test. d p ⬍ 0.05, with respect to U-2 OS, U-2/Neo8 and U-2/Neo26 cell lines, by paired t test; p ⫽ 0.054 by ANOVA test.
cells. The in vitro growth characteristics of ALP transfectants are shown in Table 2. No significant differences were observed between controls and ALP transfectants with regard to the doubling time and the clonal efficiency, whereas a significantly lower ability to grow in soft agar was found in U-2/ALP3, U-2/ALP28, and U-2/ALP40, all showing a strong positive staining for L/B/K ALP in all of the cells, but not in U-2/ALP23, displaying only a few positive cells. No significant differences were found between controls and ALP transfectants with regard to the expression of several cell surface molecules, such as integrins and adhesion molecules, apart from a significant change in the expression of ␣31 between U-2/ALP28 and controls, likely due to clonal features (Figure 1). On the other hand, a significant decrease in the MMP-9 activity (Figure 2), and in the invasive ability (Figure 3) of U-2 OS cells was observed after ALP transfection. Migratory ability was significantly reduced only in U-2/ALP40. (Figure 3).
Results
In Vivo Studies
In Vitro Studies
The presence of L/B/K ALP activity on the plasma membrane of U-2 OS osteosarcoma cells appeared to be significantly associ-
The L/B/K ALP-negative U-2 OS cell line, cotransfected with pSV2Neo and pSV2Aalp⬘ plasmids, was selected in the presence of 500 g/mL of G418, and the resulting individual neomycinresistant clones were tested for their ALP activity. Eighty-eight percent of neomycin-resistant clones exhibited measurable L/B/K ALP activity, although the levels of expression of the enzyme displayed wide variations. For this study, we chose four transfectants showing different expression and activity of L/B/K ALP (Table 1). In particular, U-2/ALP23 showed a low percentage of cells with ALP activity, a low cellular ALP activity, and a low release of ALP in the conditioned-medium; U-2/ALP3 showed a positive staining for ALP in all of the cells, a moderate cellular ALP activity, and a low release of ALP in the conditioned-medium; both U-2/ALP28 and U-2/ALP40 showed a positive staining for ALP in all of the cells, high levels of cellular ALP activity, and a high release of ALP in the conditionedmedium. The levels of L/B/K ALP activity of U-2/ALP28 and U-2/ALP40 were comparable with those found in osteoblast-like Saos-2 cells16 (Table 1), and did not significantly vary with prolonged in vitro maintenance (up to 6 months in the presence of selective medium, or up to 3 months in the absence of neomycin). U-2/Neo8 and U-2/Neo26 clones, transfected with the neo gene alone, and showing no expression of L/B/K ALP, were used as additional controls together with U-2 OS parental
Figure 1. Flow cytometric expression of some integrins and adhesion proteins in U-2 OS cells and U-2 OS ALP transfectants. Results are shown as mean ⫾ SE of three independent experiments. **p ⬍ 0.001 with respect U-2 OS, U-2/Neo8, and U-2/Neo26 cell lines, by Student’s t test.
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Figure 2. Matrix metalloproteinase-2 and MMP-9 secretion by U-2 OS cell line and its ALP transfected clones detected by ELISA in cell culture supernatants. Each bar represents the mean ⫾ SE of four independent experiments. *p ⬍ 0.05, with respect to U-2 OS, U-2/Neo8, and U-2/ Neo26 cell lines, by Student’s t test and by ANOVA test.
ated with a decrease in the tumorigenic and metastatic ability. In fact, U-2 OS parental cell line, U-2/Neo transfectants, and U-2/ALP23, which present little if any activity of L/B/K ALP, showed similar tumorigenic ability, whereas the three ALP transfectants displaying high L/B/K ALP activity at their cell surface were unable to give tumors (Table 3). Also, the experimental metastatic ability appeared to be significantly reduced in L/B/K ALP-expressing clones (Table 4), the incidence and the number of lung colonies being lower in the L/B/K ALP transfectants compared with parental cell line. Discussion Although studies concerning ALPs have been carried out over more than four decades, little is known about their biological functions. From a genetic point of view, the regulation of ALP gene is still a dilemma. Although the L/B/K ALP gene resembles the housekeeping genes in its promoter structure and widespread tissue distribution, it is not completely accurate to apply this term
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Figure 3. (a) In vitro chemotactic ability of U-2 OS ALP transfectants. A 24-h supernatant from WT-BALB/c 3T3 cells was used as a source of chemoattractant. (b) In vitro chemoinvasive ability of U-2 OS ALP transfectants through a Matrigel-coated filter. Data are expressed as mean of triplicate plates of two independent experiments ⫾ SE. *p ⬍ 0.05, **p ⬍ 0.001 with respect to U-2 OS, U-2/Neo8, and U-2/Neo26 cell lines, by Student’s t test. Analysis of variance test confirmed a significant difference between U-2/ALP40 and controls in the migration and between U-2 OS ALP transfectants and controls in the invasion (p ⬍ 0.05).
to the L/B/K ALP gene. Housekeeping implies that the gene encodes for an enzyme that performs an essential metabolic function, whereas the function for L/B/K ALP in most tissues is still unknown, the only biological role being bone mineralization, which is a tissue-specific function. Moreover, L/B/K ALP expression is different in different cell types, with a higher expression in mineralizing chondrocytes and osteoblasts. Therefore, the L/B/K ALP gene displays characteristics of both housekeeping and tissue-specific genes, and this could reflect its biological functions. In osteosarcoma, a bone-forming tumor with a heterogenous expression and activity of L/B/K ALP, we have previously observed an inverse association between the expression of this enzyme and the ability of these cells to produce tumors in athymic mice.21 Moreover, in osteosarcoma cell lines, cellular L/B/K ALP activity appears to be inversely related to the level of
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M. C. Manara et al. Alkaline phosphatase and malignancy in osteosarcoma cells
Table 3. Tumorigenic ability of U-2 OS cell line and its ALP-transfected clones Tumor growth Cell line U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40
Mice with tumor/total (%)
Mean latency (days)
Time to 5 cc volume (days)
5/8 (63%) 2/5 (40%) 4/5 (80%) 3/5 (60%) 0/5a 0/5a 0/5a
99 ⫾ 3 80 ⫾ 3 46 ⫾ 10 51 ⫾ 2 — — —
55 ⫾ 8 82 ⫾ 2 67 ⫾ 9 47 ⫾ 5 — — —
p ⬍ 0.05 with respect to U-2 OS and U-2/Neo26, by Fisher’s test.
a
expression of the hepatocyte growth factor receptor (Met/HGF).6 In particular, the presence of this receptor is associated with a higher invasive ability of sarcoma cells both in experimental6,7 and clinical conditions.20 These preliminary findings prompted us to analyze the relationship between L/B/K ALP and the malignant potential of osteosarcoma cells. In this study, we transfected U-2 OS cells, which do not present L/B/K ALP activity,23 with L/B/K ALP gene, and analyzed the in vitro and in vivo features of four clones showing different level of expression and activity of this enzyme. Apart from L/B/K ALP, we did not observe common and significant variations in the expression of other surface molecules, such as integrins and adhesion molecules, between controls and ALP-expressing clones. The only significant change was observed in the expression of ␣31 between U-2/ALP28 and controls, but this was likely due to clonal features because it was not confirmed in the other two ALP-expressing clones. No difference was also observed with regard to the doubling time and the cloning efficiency of U-2 OS variants. However, L/B/K ALP transfectants, displaying high level of expression and activity of this enzyme on their surface, showed a significantly reduced ability to grow in a semi-solid Table 4. Metastatic ability of U-2 OS cell line and its ALP transfectants
Cell line
Mice with tumor/total (%)
U-2 OS
9/9 (100%)
U-2/Neo8
5/5 (100%)
U-2/Neo26
6/9 (67%)
U-2/ALP23
10/10 (100%)
U-2/ALP3
3/10 (30%)a
U-2/ALP28
3/7 (43%)a
U-2/ALP40
4/14 (29%)a
Median (no. of metastases) 200 (20, 34, 48, 51, 200, 200, 200, 200, 200) 11 (1, 2, 11, 142, 200) 11 (0, 0, 0, 4, 11, 18, 46, 52, 124) 114 (2, 24, 27, 61, 64, 163, 171, 200, 200, 200) 0b (0, 0, 0, 0, 0, 0, 0, 2, 8, 19) 0c (0, 0, 0, 0, 15, 24, 27) 0d (0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 2, 9, 36, 66)
p ⬍ 0.05 with respect to U-2 OS and U-2/Neo8, by Fisher’s test. p ⬍ 0.001 with respect to U-2 OS, p ⬍ 0.05 with respect to U-2/Neo8, by Mann-Whitney rank sum test and by ANOVA test. c p ⬍ 0.05 with respect to U-2 OS, by Mann-Whitney rank sum test and by ANOVA test. d p ⬍ 0.001 with respect to U-2 OS, p ⬍ 0.05 with respect to U-2/Neo8, by Mann-Whitney rank sum test and by ANOVA test. a
b
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medium, to invade Matrigel, and to give pulmonary metastases in athymic mice. Also, the tumorigenic ability of U-2 OS cells appeared to be impaired by the expression and activity of L/B/K ALP. Therefore, because all of these features are mutually related parameters that are connected with malignancy,10,17 our findings suggest a loss of malignant potential of osteosarcoma cells after the induction of expression and activity of L/B/K ALP. The role of this enzyme appears to be limited to its expression at the cell surface and to its cellular activity, and to be completely independent from its release in the medium. In fact, the U-2/ ALP3 clone, which shows expression and activity of ALP in all of the cells, but a very low release of ALP in the medium, showed the same behavior of U-2/ALP28 and U-2/ALP40, which have high levels of activity both for cellular and released ALP, but differed from U-2/ALP23, which had only scattered positive cells for ALP activity. The functional consequences of L/B/K ALP activation also include a decrease in the activity of MMP-9, a metalloproteinase involved in tumor invasion and metastasis.9 Downregulation of MMP-9 activity might indeed be responsible for the reduction of aggressiveness of L/B/K ALP-expressing osteosarcoma cells. Taken together, our results suggest that cellular L/B/K ALP activity might revert the malignancy of osteosarcoma cells. In particular, likely through the downmodulation of MMP-9 activity, L/B/K ALP appears to inhibit the invasion and the metastatic ability of U-2 OS cells. Unrelated reports in different tissues indicate that L/B/K ALP might have a role in diverse biological activities, such as transendothelial transport,8 host defence,15 regulation of growth factor receptor-mediated intracellular signaling,19 or dephosphorilation of several phosphoproteins, including those containing phosphotyrosine.3,22,24 Moreover, an inverse relationship between L/B/K ALP activity and proliferation has also been suggested in endothelial cells11,18 and in Saos-2 osteoblast-like cells.14 Although this study is specifically addressed to the analysis of osteosarcoma cells, L/B/K ALP is one of the most common markers for osteoblast differentiation, and human osteosarcoma cells overexpressing this enzyme are commonly used as a model for studies on bone biology. Therefore, we believe that our findings might be indirectly useful for a better understanding of the biological role L/B/K ALP. In particular, these preliminary findings indicate that L/B/K ALP displays a wider range of activities besides bone mineralization, and appears to be involved in a number of physiological and pathological processes, such as MMPs modulation and tumor invasion and cell proliferation.
Acknowledgments: This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Italian Ministry for University and Research, the Rizzoli Institute, and the Italian Ministry of Health (Ricerca Finalizzata).
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5. Fedde, K. N. Human osteosarcoma cells spontaneously release matrix-vesiclelike structures with the capacity to mineralize. Bone Miner 17:145–151; 1992. 6. Ferracini, R., Di Renzo, M. F., Scotlandi, K., Baldini, N., Oliviero, M., Lollini, P.-L., Cremona, O., Campanacci, M., and Comoglio, P. M. The Met/HGF receptor is overexpressed in human osteosarcoma and is activated by either a paracrine or an autocrine circuit. Oncogene 10:739 –749; 1995. 7. Ferracini, R., Olivero, M., Di Renzo, M. F., Martano, M., De Giovanni, C., Nanni, P., Basso, G., Scotlandi, K., Lollini, P.-L., and Comoglio, P. M. Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas. Oncogene 12:1697–1705; 1996. 8. Harris, H. The human alkaline phosphatases: what we know and what we don’t know. Clin Chim Acta 186:133–150; 1989. 9. Himelstein, B. P., Canete-Soler, R., Bernhard, E. J., Dilks, D. W., and Muschel, R. J. Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion Metastasis 14:246 –258; 1994. 10. Laterra, J., Rosen, E., Nam, M., Ranganathan, S., Fielding, K., and Johnston, P. Scatter factor/hepatocyte growth factor expression enhances human glioblastoma tumorigenicity and growth. Biochem Biophys Res Commun 235: 743–747; 1997. 11. Lauk, S., and Trott, K. R. Endothelial cell proliferation in the rat heart following local heart irradiation. Int J Radiat Oncol Biol 57:1017–1022; 1990. 12. Lollini, P.-L., De Giovanni, C., Landuzzi, L., Nicoletti, G., and Nanni, P. Rhabdomyosarcoma cells in nude mice. In: Griffiths, J. B., Doyle, E., and Newell, D. J., Eds. Cell and Tissue Culture: Laboratory Procedure. Chichester: Wiley; 1994; module 11c:3, 1–9. 13. Malik, A. S. and Low, M. G. Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr during butanol extraction. An investigation of the role of endogenous phospho-inositide-specific phospholipases. Biochem J 240:519 –527; 1986. 14. Mohan, S., Strong, D. D., Hilliker, S., Malpe, K. L., Farley, J., and Baylink, D. J. Dibutyryl cyclic adenosine monophosphate differentially regulates cell proliferation in low and high alkaline phosphatase SaOS-2 human osteosarcoma cells: evidence for mediation by the insuline-like growth factor-II system. J Cell Physiol 156:462– 468; 1993. 15. Poelstra, K., Bakker, W. W., Klok, P. A., Kamps, J. A. A. M., Hardonk, M. J., and Meijer, D. K. F. Dephosphorylation of endotoxin by alkaline phosphatase in vivo. Am J Pathol 151:1163–1169; 1997. 16. Rodan, S. B., Imai, Y., Thiede, M. A., Wesolowski, G., Thompson, D., Bar-Shaviz, Z., Shull, S., Mann, K., and Rodan, G. Characterization of a human osteosarcoma cell line (SaOS-2) with osteoblastic properties. Cancer Res 47:4961– 4966; 1987. 17. Sapi, E., Flick, M. B., Rodov, S., Gilmore, H. M., Kelley, M., Rockwell, S., and Kacinski, B. M. Independent regulation of invasion and anchorage-independent growth by different autophosphorylation. Cancer Res 56:5704 –571; 1996.
Bone Vol. 26, No. 3 March 2000:215–220 18. Schultz-Hector, S. and Haghayegh, S. b-FGF expression in human and murine squamous cell carcinomas (ssc) and its relationship to regional endothelial cell proliferation. Cancer Res 53:1444 –1449; 1993. 19. Schultz-Hector, S., Balz, K., Bo¨hm, M., Ikehara, Y., and Rieke, L. Cellular localization of endothelial alkaline phosphatase reaction product and enzyme protein of the miocardium. J Histochem Cytochem 41:1813–1821; 1993. 20. Scotlandi, K., Baldini, N., Olivero, M., Di Renzo, M. F., Martano, M., Serra, M., Manara, M. C., Comoglio, P. M., and Ferracini, R. Expression of Met/ hepathocyte growth factor receptor gene and malignant behavior of musculoskeletal tumors. Am J Pathol 149:1209 –1219; 1996. 21. Scotlandi, K., Serra, M., Manara, M. C., Nanni, P., Nicoletti, G., Landuzzi, L., Maurici, D., and Baldini, N. Human osteosarcoma cells, tumorigenic in nude mice, express 1 integrins and low levels of alkaline phosphatase activity. Int J Oncol 3:963–969; 1993. 22. Sparks, J. W. and Brautigan, D. L. Specificity of protein phosphotyrosine phosphatases. J Biol Chem 260:2042–2045; 1984. 23. Stinson, R. A., Thacker, J. D., and Lin, C. C. Expression and nature of the alkaline phosphatase gene in cultured osteosarcoma cells. Clin Chim Acta 221:105–114; 1993. 24. Swarup, G., Cohen, S., and Garbers, D. L. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J Biol Chem 256:8197– 8201; 1981. 25. Weiss, M. J., Ray, K., Henthorn, P. S., Lamb, B., Kadesch, T., and Harris, H. Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 263:12002–12010; 1988. 26. Weiss, M. V., Cole, E. C., Ray, K., Whyte, M. P., Lafferty, M. A., Mulivor, R. A., and Harris, H. A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia. Proc Natl Acad Sci USA 85:7666 –7669; 1988. 27. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16:777–785; 1979. 28. Yoon, K., Golub, E., and Rodan, G. A. Alkaline phosphatase cDNA transfected cells promote calcium and phosphate deposition. Connect Tissue Res 22:17– 25; 1989.
Date Received: June 10, 1999 Date Revised: November 12, 1999 Date Accepted: November 15, 1999
ORIGINAL ARTICLES
Reversal of Malignant Phenotype in Human Osteosarcoma Cells Transduced With the Alkaline Phosphatase Gene M. C. MANARA,1 N. BALDINI,1 M. SERRA,1 P.-L. LOLLINI,2 C. De GIOVANNI,2 M. VACCARI,3 A. ARGNANI,3 S. BENINI,1 D. MAURICI,1 P. PICCI,1 and K. SCOTLANDI1 1
Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy Istituto di Cancerologia, Universita` degli Studi di Bologna, Bologna, Italy 3 IST, Istituto di Ricerche per la Ricerca sul Cancro-Genova, Unita` Satellite di Biotecnologie, Bologna, Italy 2
in fundamental biological processes. However, as yet, the physiological role of L/B/K ALP is unknown, except that the bone isoenzyme is thought to play an essential role in normal skeletal mineralization. Bone ALP is anchored to the outer plasma membrane of osteoblasts by a proteoglycan-inositol linkage to phosphatidylinositol8 and can be released from the cell surface by a glycan-inositol hydrolase and/or surrounded by membrane vesicles.5,13 Evidence for a role of bone ALP in mineralization derives from the fact that the introduction of bone ALP cDNA into ALP-negative cells confers an in vitro capacity for mineralization,28 and that missense mutations of bone ALP gene are associated with a lethal disease characterized by osteomalacia and a deficiency of L/B/K ALP in all tissues (placental and intestinal ALP levels being unaffected).26 However, the precise biochemical function of bone ALP activity is still unknown, and its role in normal bone physiology is not completely understood. Even less defined is its clinical significance in osteosarcoma, a bone-forming neoplasm showing a wide range of expression of ALP. With regard to osteosarcoma, we have previously demonstrated the existence of an inverse correlation between cellular bone ALP expression and tumor aggressiveness.21 In this paper, we further investigated this relationship by transfecting the L/B/K ALP gene25 in an osteosarcoma cell line with no evidence of basal expression of this enzyme.23 The in vitro and in vivo behavior of selected transfectants were analyzed with regard to their invasive, metastatic, and tumorigenic ability.
Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the boneforming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme. (Bone 26:215–220; 2000) © 2000 by Elsevier Science Inc. All rights reserved. Key Words: Alkaline phosphatase; Osteosarcoma; Matrix metalloproteinase; Tumorigenicity; Metastasis; Athymic mice. Introduction
Material and Methods
Alkaline phosphatases (ALP) (orthophosphoric-monoester phosphohydrolase) are a group of cell surface glycoproteins that hydrolize a broad range of monophosphate esters at an optimum alkaline pH. Based on heat stability, sensitivity to inhibitors, antigenicity, and electrophoretic mobility, four major ALP isoenzymes have been characterized so far,8 including placental, intestinal, liver/bone/kidney (L/B/K), and germ cell ALP, each encoded by separate genes. Each form of human ALP exhibits a characteristic pattern of tissue distribution, placental and intestinal ALP being found predominantly in the tissues for which they are named, whereas L/B/K ALP is widely distributed. Alkaline phosphatases are found in organisms ranging from bacteria to mammals, and their conserved presence suggests an involvement
Cell Lines U-2 OS human osteosarcoma cells were routinely cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with penicillin (100 U/mL), streptomycin (100 g/mL) (Life Technologies, Paisley, Scotland), and 10% heat-inactivated fetal calf serum (FCS; Biological Industries, Kibbutz Beth Haemek, Israel). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. The transfection of U-2 OS cell line was performed by calcium phosphate coprecipitation 24 h after seeding of 105 cells in a 100-mm dish.27 Cells were cotransfected with 10 g of pSV2Aalp⬘, an expression vector containing the full-lenght L/B/K ALP cDNA26 and 10 g of pSV2Neo, containing the neo gene, which confers resistance to neomycin. After 24 h of DNA exposure, medium was removed and cells were maintained in normal growth medium for 2 more days. Cells were then subjected to selective medium containing 500 g/mL of G418
Address for correspondence and reprints: Dr.ssa K. Scotlandi, Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via Di Barbiano 1/10, 40126 Bologna, Italy. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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(Sigma, St. Louis, MO) and cultured for 2 weeks with a selective medium change every 2 days. Individual colonies were cloned with glass cylinders and expanded in selective medium. U-2/ALP3, U-2/ALP23, U-2/ALP28, and U-2/ALP40 are representative of ALP cotransfected clones and were chosen for their different range of ALP activity. To obtain additional controls, U-2 OS cells were transfected with pSV2Neo alone and selected in medium containing G418 (500 g/mL). U-2/Neo8 and U-2/Neo26 are representative of this series of transfectants.
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S.A., Marseilles, France), 1:10 dilution; GC-4 (anti-A-CAM; Sigma), 1:100 dilution; CDw49b VLA2 (anti-alpha 2 chain, ␣21; Immunotech S.A.), 1:10 dilution; P1B5 (anti-alpha3 chain, ␣31; Oncogene Research Product) 1:20 dilution; CDw49d VLA4 (anti-alpha 4 chain, ␣41; Immunotech S.A.), 1:10 dilution; CDw49e VLA5 (anti-alpha 5 chain, ␣51; Immunotech S.A.), 1:10 dilution; and CDw49f VLA6 (anti-alpha 6 chain, ␣61; Immunotech S.A.), 1:10 dilution. Chemotaxis Assay
In Vitro Growth Features For the determination of population doubling time, 0.5 ⫻ 106 cells were seeded in IMDM plus 10% FCS in 25-cm2 flasks (Falcon, Oxnard, CA) and harvested daily by using 0.25% trypsin/0.002% EDTA (Life Technologies, Paisley, Scotland). Doubling time was calculated during the logarithmic growth phase. Cell viability was determined by Trypan blue dye exclusion. For the evaluation of cloning efficiency, 200 –1600 cells were seeded in 60-mm dishes. After 7 days, cells were fixed with methanol for 10 min at room temperature, and stained with Giemsa. Colonies of at least 10 cells were counted using an inverted microscope. Soft-Agar Assay Anchorage-independent growth was determined in 0.33% agarose (FMC Bio Products, Rockland, ME) with a 0.5% agarose underlay. Cell suspensions (10,000 –33,000 cells per 60-mm dish) were plated in a semi-solid medium (IMDM with 10% FCS containing 0.33% agarose). Colonies were counted after 14 days. ALP Activity The percentage of cells displaying ALP activity at their cell surface was evaluated on cytospins obtained 4 days after cell seeding using a cytochemical method (Kit 86R; Sigma, St. Louis, MO). The percentage of positive cells was calculated on at least 300 cells. Cellular ALP activity was also analyzed 4 days after seeding. Approximately 106 cells were resuspended in homogenization buffer (1 mmol/L MgCl2, 1 mmol/L CaCl2, 20 mol/L ZnCl2, 0.1 mol/L NaCl, 0.1% Triton X-100, 0.05 mol/L TrisHCl, pH 7.4) and disrupted by pipetting. The homogenate was used for the ALP assay, which was performed with p-nitrophenol phosphate as a substrate, according to the instructions of the manufacturer (Sigma). L/B/K ALP activity was normalized for the content of protein in the sample. Proteins were measured according to the Bradford method2 using bovine serum albumin (BSA) fraction V (Sigma) as a standard. In particular, one unit of L/B/K ALP activity is defined as the amount of enzyme capable of transforming 1 mol of substrate in 1 min at 25°C. L/B/K ALP activity in the conditioned medium due to the released ALP was also measured 4 days after cell seeding using p-nytrophenyl phosphate as a substrate, in accordance to the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany). Phenotypic Characteristics The expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-3 (LFA-3), and A-cell adhesion molecule (A-CAM) as well as of ␣21, ␣31, ␣41, ␣51, and ␣61 integrins was determined by flow cytometry after indirect immunofluorescence using the following monoclonal antibodies: LFA-3 (anti-LFA-3; Immunotech S.A., Marseille, France), 1:20 dilution; CD54 ICAM (anti-ICAM-1; Immunotech
The chemotaxis assay was made using Transwell chambers (Costar, Cambridge, MA) as previously described.4 Briefly, 1.5 ⫻ 105 cells were resuspended in Dulbecco’s modified medium (Life Technologies, St. Louis, MO) containing 0.1% BSA (Sigma) and seeded in the upper compartment of the chamber. A 24-h supernatant from BALB/c 3T3 cells collected in the absence of serum was placed in the lower compartment as a source of chemoattractant. The two compartments were separated by an 8.0-m pore size, polyvinylpirrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA) coated with 5 g/mL gelatin (Sigma). Cells were allowed to migrate for 6 h at 37°C. After incubation, cells that had migrated to the lower side of the filter were fixed in ethanol and stained with toluidine blue. Five to 10 fields/filter were counted at ⫻160. Three different experiments were made for each cell line. Chemoinvasion Assay This assay is a modification of the chemotaxis assay.1 Polycarbonate filters coated with Matrigel (16.5 g/filter; Collaborative Biomedical Products, Bedford, MA) were placed in chemotaxis chambers. After 6 h of incubation, cells on the lower surface of filter were scored as for chemotaxis. Three different experiments were made for each cell line. Detection of Matrix Metalloproteinase-2 and -9 Activity Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated on confluent monolayers of U-2 OS cells and ALP transfectants by replacing the culture medium with fresh serumfree medium. Supernatants were collected 24 h later and, to evaluate MMP-9 activity, were concentrated with a Centricon Plus-20 centrifugal filter devise provided with an ultrafiltration membrane with a 30,000 nominal molecular weight cut-off limit (Millipore Corporation, Bedford, MA). Matrix metalloproteinase-2 and 9 activity was determined by the enzyme-linked immunosorbent assay (ANOVA) test Biotrak MMP-2 or MMP-9 activity assay systems (Amersham Pharmacia Biotech, Milan, Italy). The test was performed according to the manufacturer’s instructions. Briefly, standards and samples were incubated at 4°C overnight in microtiter wells precoated with anti-MMP-2 or anti-MMP-9 antibody. After extensive washings, any bound MMP-2 or MMP-9 was activated by adding aminophenylmercuric acetate. Active MMP-2 or MMP-9 was then detected through the activation of a modified urokinase proenzyme and the subsequent cleavage of its chromogenic peptide substrate. The resultant color was read at 405 nm, and the concentration of active MMP-2 or MMP-9 was determined by interpolation from the standard curve. In Vivo Growth Female 4 –5-week-old Cr1:nu/nu (CD-1)BR athymic mice (Charles River Italia, Como, Italy) were used. Tumorigenicity was determined after subcutaneous injection of 30 ⫻ 106 cells.
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Table 1. In vitro ALP activity of U-2 OS cell line and ALP-transfected clones measured 4 days after cell seeding
Cell line
Expression of membrane-bound ALP (% of positive cells/total)
Membrane-bound ALP activity (mU/mg protein)
ALP activity in conditioned medium (mU/ mL/106 cells)
0 0 0 15 ⫾ 7 100 100 100 100
0 0 0 14.5 ⫾ 0.5 46.0 ⫾ 5.0 105.5 ⫾ 5.5 81.5 ⫾ 12.5 104.7 ⫾ 18.4
0.2 0.3 0.2 4.4 ⫾ 1.6 4.2 ⫾ 0.8 102.7 ⫾ 18.7 136.5 ⫾ 38.7 120.8 ⫾ 20.8
U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40 Saos-2
Tumor growth was assessed once weekly by measuring tumor volume, calculated as /6 ⫻ [公(ab)]3, where a and b are the two major diameters.12 Mice were killed and autopsied when mean tumor volume was 5 cc or, in mice with no evidence of tumor, at 6 months after cell inoculation. Histological sections obtained from the tumors growing in athymic mice were stained with hematoxylin-eosin. Experimental metastatic ability was evaluated after injection of 2 ⫻ 106 cells in a tail lateral vein. To obtain natural killer-depressed animals, mice were injected intravenously with 0.4 mL of a 1:25 dilution of anti-asialo GM1 antiserum (Wako, Du¨sseldorf, Germany) 24 h before cell inoculation. Two months later, mice were killed and the number of pulmonary metastases was determined by counting with a stereomicroscope after staining with black India ink. Statistical Analysis Student’s t test and ANOVA were used for repeated measurement values. For frequency data, Fisher’s, Mann-Whitney, and ANOVA rank sum tests were used.
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Table 2. In vitro growth parameters of U-2 OS cell line and ALPtransfected clones
Cell line
Doubling time (h)
Cloning efficiencya (%)
No. of colonies in soft agarb
18.3 ⫾ 0.2 19.2 ⫾ 1.2 21.9 ⫾ 3.6 24.1 ⫾ 6.7 23.5 ⫾ 4.1 27.1 ⫾ 4.6 26.1 ⫾ 5.8
59.7 ⫾ 1.7 55.7 ⫾ 5.8 52.5 ⫾ 3.1 51.9 ⫾ 2.8 50.1 ⫾ 4.2 58.4 ⫾ 1.9 50.6 ⫾ 0.4c
648 ⫾ 55 540 ⫾ 20 657 ⫾ 55 495 ⫾ 20 324 ⫾ 7d 391 ⫾ 7d 316 ⫾ 51d
U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40 a
Cloning efficiency was determined by counting the colonies 1 week after seeding with 200 –1,600 cells/cm2. Data are expressed as mean of triplicate plates ⫾ SE. b 10,000 cells were plated in 0.33% agarose with IMDM plus 10% FCS. Data are expressed as mean of triplicate plates ⫾ SE. c p ⬍ 0.05, with respect to U-2 OS cell line, by paired t test. d p ⬍ 0.05, with respect to U-2 OS, U-2/Neo8 and U-2/Neo26 cell lines, by paired t test; p ⫽ 0.054 by ANOVA test.
cells. The in vitro growth characteristics of ALP transfectants are shown in Table 2. No significant differences were observed between controls and ALP transfectants with regard to the doubling time and the clonal efficiency, whereas a significantly lower ability to grow in soft agar was found in U-2/ALP3, U-2/ALP28, and U-2/ALP40, all showing a strong positive staining for L/B/K ALP in all of the cells, but not in U-2/ALP23, displaying only a few positive cells. No significant differences were found between controls and ALP transfectants with regard to the expression of several cell surface molecules, such as integrins and adhesion molecules, apart from a significant change in the expression of ␣31 between U-2/ALP28 and controls, likely due to clonal features (Figure 1). On the other hand, a significant decrease in the MMP-9 activity (Figure 2), and in the invasive ability (Figure 3) of U-2 OS cells was observed after ALP transfection. Migratory ability was significantly reduced only in U-2/ALP40. (Figure 3).
Results
In Vivo Studies
In Vitro Studies
The presence of L/B/K ALP activity on the plasma membrane of U-2 OS osteosarcoma cells appeared to be significantly associ-
The L/B/K ALP-negative U-2 OS cell line, cotransfected with pSV2Neo and pSV2Aalp⬘ plasmids, was selected in the presence of 500 g/mL of G418, and the resulting individual neomycinresistant clones were tested for their ALP activity. Eighty-eight percent of neomycin-resistant clones exhibited measurable L/B/K ALP activity, although the levels of expression of the enzyme displayed wide variations. For this study, we chose four transfectants showing different expression and activity of L/B/K ALP (Table 1). In particular, U-2/ALP23 showed a low percentage of cells with ALP activity, a low cellular ALP activity, and a low release of ALP in the conditioned-medium; U-2/ALP3 showed a positive staining for ALP in all of the cells, a moderate cellular ALP activity, and a low release of ALP in the conditioned-medium; both U-2/ALP28 and U-2/ALP40 showed a positive staining for ALP in all of the cells, high levels of cellular ALP activity, and a high release of ALP in the conditionedmedium. The levels of L/B/K ALP activity of U-2/ALP28 and U-2/ALP40 were comparable with those found in osteoblast-like Saos-2 cells16 (Table 1), and did not significantly vary with prolonged in vitro maintenance (up to 6 months in the presence of selective medium, or up to 3 months in the absence of neomycin). U-2/Neo8 and U-2/Neo26 clones, transfected with the neo gene alone, and showing no expression of L/B/K ALP, were used as additional controls together with U-2 OS parental
Figure 1. Flow cytometric expression of some integrins and adhesion proteins in U-2 OS cells and U-2 OS ALP transfectants. Results are shown as mean ⫾ SE of three independent experiments. **p ⬍ 0.001 with respect U-2 OS, U-2/Neo8, and U-2/Neo26 cell lines, by Student’s t test.
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Figure 2. Matrix metalloproteinase-2 and MMP-9 secretion by U-2 OS cell line and its ALP transfected clones detected by ELISA in cell culture supernatants. Each bar represents the mean ⫾ SE of four independent experiments. *p ⬍ 0.05, with respect to U-2 OS, U-2/Neo8, and U-2/ Neo26 cell lines, by Student’s t test and by ANOVA test.
ated with a decrease in the tumorigenic and metastatic ability. In fact, U-2 OS parental cell line, U-2/Neo transfectants, and U-2/ALP23, which present little if any activity of L/B/K ALP, showed similar tumorigenic ability, whereas the three ALP transfectants displaying high L/B/K ALP activity at their cell surface were unable to give tumors (Table 3). Also, the experimental metastatic ability appeared to be significantly reduced in L/B/K ALP-expressing clones (Table 4), the incidence and the number of lung colonies being lower in the L/B/K ALP transfectants compared with parental cell line. Discussion Although studies concerning ALPs have been carried out over more than four decades, little is known about their biological functions. From a genetic point of view, the regulation of ALP gene is still a dilemma. Although the L/B/K ALP gene resembles the housekeeping genes in its promoter structure and widespread tissue distribution, it is not completely accurate to apply this term
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Figure 3. (a) In vitro chemotactic ability of U-2 OS ALP transfectants. A 24-h supernatant from WT-BALB/c 3T3 cells was used as a source of chemoattractant. (b) In vitro chemoinvasive ability of U-2 OS ALP transfectants through a Matrigel-coated filter. Data are expressed as mean of triplicate plates of two independent experiments ⫾ SE. *p ⬍ 0.05, **p ⬍ 0.001 with respect to U-2 OS, U-2/Neo8, and U-2/Neo26 cell lines, by Student’s t test. Analysis of variance test confirmed a significant difference between U-2/ALP40 and controls in the migration and between U-2 OS ALP transfectants and controls in the invasion (p ⬍ 0.05).
to the L/B/K ALP gene. Housekeeping implies that the gene encodes for an enzyme that performs an essential metabolic function, whereas the function for L/B/K ALP in most tissues is still unknown, the only biological role being bone mineralization, which is a tissue-specific function. Moreover, L/B/K ALP expression is different in different cell types, with a higher expression in mineralizing chondrocytes and osteoblasts. Therefore, the L/B/K ALP gene displays characteristics of both housekeeping and tissue-specific genes, and this could reflect its biological functions. In osteosarcoma, a bone-forming tumor with a heterogenous expression and activity of L/B/K ALP, we have previously observed an inverse association between the expression of this enzyme and the ability of these cells to produce tumors in athymic mice.21 Moreover, in osteosarcoma cell lines, cellular L/B/K ALP activity appears to be inversely related to the level of
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Table 3. Tumorigenic ability of U-2 OS cell line and its ALP-transfected clones Tumor growth Cell line U-2 OS U-2/Neo8 U-2/Neo26 U-2/ALP23 U-2/ALP3 U-2/ALP28 U-2/ALP40
Mice with tumor/total (%)
Mean latency (days)
Time to 5 cc volume (days)
5/8 (63%) 2/5 (40%) 4/5 (80%) 3/5 (60%) 0/5a 0/5a 0/5a
99 ⫾ 3 80 ⫾ 3 46 ⫾ 10 51 ⫾ 2 — — —
55 ⫾ 8 82 ⫾ 2 67 ⫾ 9 47 ⫾ 5 — — —
p ⬍ 0.05 with respect to U-2 OS and U-2/Neo26, by Fisher’s test.
a
expression of the hepatocyte growth factor receptor (Met/HGF).6 In particular, the presence of this receptor is associated with a higher invasive ability of sarcoma cells both in experimental6,7 and clinical conditions.20 These preliminary findings prompted us to analyze the relationship between L/B/K ALP and the malignant potential of osteosarcoma cells. In this study, we transfected U-2 OS cells, which do not present L/B/K ALP activity,23 with L/B/K ALP gene, and analyzed the in vitro and in vivo features of four clones showing different level of expression and activity of this enzyme. Apart from L/B/K ALP, we did not observe common and significant variations in the expression of other surface molecules, such as integrins and adhesion molecules, between controls and ALP-expressing clones. The only significant change was observed in the expression of ␣31 between U-2/ALP28 and controls, but this was likely due to clonal features because it was not confirmed in the other two ALP-expressing clones. No difference was also observed with regard to the doubling time and the cloning efficiency of U-2 OS variants. However, L/B/K ALP transfectants, displaying high level of expression and activity of this enzyme on their surface, showed a significantly reduced ability to grow in a semi-solid Table 4. Metastatic ability of U-2 OS cell line and its ALP transfectants
Cell line
Mice with tumor/total (%)
U-2 OS
9/9 (100%)
U-2/Neo8
5/5 (100%)
U-2/Neo26
6/9 (67%)
U-2/ALP23
10/10 (100%)
U-2/ALP3
3/10 (30%)a
U-2/ALP28
3/7 (43%)a
U-2/ALP40
4/14 (29%)a
Median (no. of metastases) 200 (20, 34, 48, 51, 200, 200, 200, 200, 200) 11 (1, 2, 11, 142, 200) 11 (0, 0, 0, 4, 11, 18, 46, 52, 124) 114 (2, 24, 27, 61, 64, 163, 171, 200, 200, 200) 0b (0, 0, 0, 0, 0, 0, 0, 2, 8, 19) 0c (0, 0, 0, 0, 15, 24, 27) 0d (0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 2, 9, 36, 66)
p ⬍ 0.05 with respect to U-2 OS and U-2/Neo8, by Fisher’s test. p ⬍ 0.001 with respect to U-2 OS, p ⬍ 0.05 with respect to U-2/Neo8, by Mann-Whitney rank sum test and by ANOVA test. c p ⬍ 0.05 with respect to U-2 OS, by Mann-Whitney rank sum test and by ANOVA test. d p ⬍ 0.001 with respect to U-2 OS, p ⬍ 0.05 with respect to U-2/Neo8, by Mann-Whitney rank sum test and by ANOVA test. a
b
219
medium, to invade Matrigel, and to give pulmonary metastases in athymic mice. Also, the tumorigenic ability of U-2 OS cells appeared to be impaired by the expression and activity of L/B/K ALP. Therefore, because all of these features are mutually related parameters that are connected with malignancy,10,17 our findings suggest a loss of malignant potential of osteosarcoma cells after the induction of expression and activity of L/B/K ALP. The role of this enzyme appears to be limited to its expression at the cell surface and to its cellular activity, and to be completely independent from its release in the medium. In fact, the U-2/ ALP3 clone, which shows expression and activity of ALP in all of the cells, but a very low release of ALP in the medium, showed the same behavior of U-2/ALP28 and U-2/ALP40, which have high levels of activity both for cellular and released ALP, but differed from U-2/ALP23, which had only scattered positive cells for ALP activity. The functional consequences of L/B/K ALP activation also include a decrease in the activity of MMP-9, a metalloproteinase involved in tumor invasion and metastasis.9 Downregulation of MMP-9 activity might indeed be responsible for the reduction of aggressiveness of L/B/K ALP-expressing osteosarcoma cells. Taken together, our results suggest that cellular L/B/K ALP activity might revert the malignancy of osteosarcoma cells. In particular, likely through the downmodulation of MMP-9 activity, L/B/K ALP appears to inhibit the invasion and the metastatic ability of U-2 OS cells. Unrelated reports in different tissues indicate that L/B/K ALP might have a role in diverse biological activities, such as transendothelial transport,8 host defence,15 regulation of growth factor receptor-mediated intracellular signaling,19 or dephosphorilation of several phosphoproteins, including those containing phosphotyrosine.3,22,24 Moreover, an inverse relationship between L/B/K ALP activity and proliferation has also been suggested in endothelial cells11,18 and in Saos-2 osteoblast-like cells.14 Although this study is specifically addressed to the analysis of osteosarcoma cells, L/B/K ALP is one of the most common markers for osteoblast differentiation, and human osteosarcoma cells overexpressing this enzyme are commonly used as a model for studies on bone biology. Therefore, we believe that our findings might be indirectly useful for a better understanding of the biological role L/B/K ALP. In particular, these preliminary findings indicate that L/B/K ALP displays a wider range of activities besides bone mineralization, and appears to be involved in a number of physiological and pathological processes, such as MMPs modulation and tumor invasion and cell proliferation.
Acknowledgments: This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Italian Ministry for University and Research, the Rizzoli Institute, and the Italian Ministry of Health (Ricerca Finalizzata).
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Date Received: June 10, 1999 Date Revised: November 12, 1999 Date Accepted: November 15, 1999