Phenotypic characterization of chondrosarcoma-derived cell lines

Cancer Letters 226 (2005) 143–154 www.elsevier.com/locate/canlet

Phenotypic characterization of chondrosarcoma-derived cell lines* C.M. Scho¨rlea, F. Fingera, A. Zienb, J.A. Blockc, P.M. Gebharda, T. Aignera,* a

Osteoarticular and Arthritis Research, Department of Pathology, University of Erlangen-Nu¨rnberg, Krankenhausstr. 8-10, D-91054 Erlangen, Germany b Max Planck Institute for Biological Cybernetics, Tuebingen, Germany c Rheumatology, Rush University Medical Center, Chicago, IL, USA Received 27 August 2004; received in revised form 4 November 2004; accepted 9 November 2004

Abstract Gene expression profiling of three chondrosarcoma derived cell lines (AD, SM, 105 KC) showed an increased proliferative activity and a reduced expression of chondrocytic-typical matrix products compared to primary chondrocytes. The incapability to maintain an adequate matrix synthesis as well as a notable proliferative activity at the same time is comparable to neoplastic chondrosarcoma cells in vivo which cease largely cartilage matrix formation as soon as their proliferative activity increases. Thus, the investigated cell lines are of limited value as substitute of primary chondrocytes but might have a much higher potential to investigate the behavior of neoplastic chondrocytes, i.e. chondrosarcoma biology. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chondrocyte; Cell line; Expression analysis; Functional genomics

1. Introduction

Abbreviations ADAMTS, a disintegrin and metalloproteinase with thrombospondin type 1 motif; Col, collagen; IL-1b, interleukin 1b; MMP, matrix metalloproteinases; OA, osteoarthritic; qPCR, quantitative PCR; TIMP, tissue inhibitor of metalloproteinases. * Supported by the Federal Ministry of Education and Research (BMBF) and the Interdisciplinary Center of Clinical Research (IZKF) of the University Hospital of the University of ErlangenNu¨rnberg, the Ministry of Research (grant 01GG9824). * Corresponding author. Tel.: C49 9131 8522857; fax: C49 9131 8524745. E-mail address: [email protected] (T. Aigner).

Primary chondrocyte cultures isolated from young animals have been widely used to investigate basic biological features of chondrocytes as they are easily obtained and maintain a cartilage-specific phenotype in vitro [1]. Investigations of primary adult human chondrocytes, however, are much more limited, because the gain of a sufficient number of chondrocytes from operative procedures is limited and the biological and functional characteristics of primary chondrocytes differ much in between different donors [1]. Also, proliferative activity, however, implicates

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.11.022

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dedifferentiation with loss of the typical chondrocytic cell morphology as well as a significant change in the pattern of gene expression [2]. Therefore, long term investigations of physiological characteristics of primary chondrocytes as well as chondrocytic alterations in the development of osteoarthritis are hampered due to the quick loss of phenotypic stability. Thus, chondrocytic cell lines are often used as a substitute for primary chondrocytes. In this situation, chondrosarcoma derived cell lines suggest to be a promising substitute with sufficient proliferative activity and the ability to display a consistent response to phenotype modulating stimuli [1,3–5]. Chondrosarcoma derived cell lines were so far predominantly analyzed for selected genes [6–9] whereas an extensive gene profiling and a correlation of the gene expression pattern to primary chondrocytes is missing. The following study investigated the gene expression profiles of three chondrosarcoma derived cell lines using the cDNA array technology. Quantitative PCR was used to confirm characteristic findings of selected genes. The expression profiles finally were correlated with reference data from primary adult chondrocytes in order to analyze the suitability of chondrosarcoma derived cell lines as substitutes of primary articular chondrocytes for experimental research.

tissue-culture dishes at 37 8C at normal oxygen concentration in the presence of 5% CO2. Medium was changed every three days. Cells were cultured in monolayers (w0.5!106 cells per Petri-dish) and grown to confluence. Cells were mechanically disrupted from the dishes and stripped of their matrix by digestion with 0.25% trypsin, washed twice with PBS, and concentrated with low-speed centrifugation. For culturing in alginate beads, confluent monolayer cultures were treated with trypsin-EDTA, washed with PBS, and resuspended in a filtersterilized solution of 1.2% (w/v) alginate (Kelgin LV, Kelco Inc., San Diego, USA) in 0.15 M NaCl at a density of 4!106 cells/ml according to the protocol of Ha¨uselmann et al. [11]. Beads formed after dropwise addition into 100 mM CaCl2 which encapsulated the cells (about 4!104 cells/bead). After washing in 0.15 M NaCl, the alginate beads were cultured in Petri-dishes for 10 days. Cells were recovered from the alginate by depolymerising the alginate in three volumes of 55 mM Na citrate/0.15 M NaCl at 37 8C for 10 min. The cells were pelleted by cytocentrifugation and washed with PBS. The cell number and viability of cells was checked microscopically in a hemocytometer with staining of 0.4% Trypan blue (w/v) in PBS.

2. Material and methods

2.2. Cell isolation and stimulation of primary articular chondrocytes

2.1. Cell lines—culture in monolayer and alginate beads All cell lines were gained from tumors obtained at resection and were established as described before [10]. The origin and the pathohistological classification of the different tumors are listed in Table 1. The protocol for the cell line generation has been described in detail previously [10]. Cells were cultured in a medium consisting of 40% of Dulbecco’s modified Eagle medium (DMEM), 40% minimum essential medium, alpha modification (Gibco Laboratories, New York) supplemented with 10% Ham’s F12 Medium, 10% fetal bovine serum with 100 nM hydrocortisone, 100 ng/ml of insulin, 25 mg/ml of ascorbate and 0.05 mg/ml gentamycin (Omega Scientific, Tarzana, CA). The cells are cultured in coated

Normal human knee articular cartilage was obtained from a 48-years old male donor at autopsy, within 48 h of death. For PCR validation experiments, cells were isolated from two additional donors with normal articular cartilage (71 years, male; 59 years, male) as described previously [12]. Chondrocytes Table 1 Clinicopathologic features of cell lines AD, SM, 105 KC [6,7] Cell line

Sex/e

Site

Grade

Subtype

AD

56 y/o female 36 y/o male 66 y/o male

Right pelvis

II

Left femur

III

Left femur

II

Conventional chondrosarcoma Conventional chondrosarcoma Conventional chondrosarcoma

SM 105 KC

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145

Table 2 Sequences of primers and probes for quantitative online-PCR experiments Acc. no.

Primer

nM

Probe

Mg (mM)

GAPDH

NM_002046

5.5

M10277

TTTCGTGGATGCCACGACTCCAT

6

aggrecan

NM_013227

CCATGCAATTTGAACTGGCGCC

6

COL1A1

NM_000088

AATCACCTGCGTACAACGGCCTCA

6,5

COL2A1

NM_001844

ACCTTCCTACGCCTGCTGTCCACG

5,5

COL2A1 (Splice variant A) Col2-2.exon

L10347

50 900 900 50 50 50 50 300 50 300 300

CACTTCCCGTTCTCCC

b-actin

fw: GAGTGAGTCGGTC rv: GAATGGTGATGGGATTTC fw: GCCCTGGCACTCTTCCA rv: TTGCGGATGTCCACGTCA fw:ACTTCCGCTGGTCATGGA rv: TCTCGTGCCATCATCACC fw: GGCCAACGAACATC rv: ATCACGTCATCGCACAACA fw:CAACACTGCCAACGTCCAT rv: CTGCTTCGTCCATGCAAT fw: GTATAATGATAGATGTGTGGA

CACACACATCCGGCGGC

6

COL10A1

NM_000493

300 300

ACGCTGAACGATACCAAACGCCCAC

5,5

MMP-1

NM_002421

rv: GTCGTCGCGACTCC fw: TGCTTATCCTTGAACTTGGTTCAT rv: CTGTGTCTTGGTGTTGGGTTG fw: CTGTTCGGACAATGTGCT

ACGGATACCCCAGACATCTACCTCC

6.5

MMP-3

NM_002422

4

NM_002427

900 900 50 900

AACTTCATATGCGGCATCCACGCC

MMP-13

rv: TCGATATGCTTCACTTCTGG fw: TTTTGGCCATCTCTTCCTTCA rv: TGTGGATGCCTCTTGGGTATC fw: TCCTCTTCTTGCTGGACTCATT

TCCTCACAAATCATCTTCATCACCACCAC

7

MMP-14

NM_004995

rv: CGCTCTGCAAACTGGGTC fw: TGCCTGCGTCCATCAACACT

50 900

AACGAATTTGCCATCCTTCCTCTCGT

7

ADAMTS-1

NM_006988

7

AF148213

ACTGCCCATCCATTGTCCGA

6

ADAMTS-5

AF142099

ATGGCCACCAACACAACCC

4.5

CDRAP

NM_006533

CCGACTGCCGATTCCTGACCATTC

6,5

SOX9

Z46629

50 900 900 900 50 300 900 300 900 900 50

CGTTTTGCCCCAGTTGTATGGT

ADAMTS-4

rv: CATCAAACACCCAATGCTTGTC fw: GCCAAGCATTGGCTACTTC rv: GTGGAATCTGGGCTACATGGA fw: TGCCCGCTTCATCACTGA rv: CAATGGCCTCTGGTTTGTC fw: CGCTGCCACCACACTCAA rv: CGTTGCTCCTCATGGTCATCT fw: GCTGTGGCCCTTCGACTA rv: AATCTCCCTGAACGCTGCCT fw: ACACACCTCACTCGACCTTG rv: GGAATTCTGGTTGGTCCTCTCTT

TTGATCATCTCGGCCATCGTCGC

7

were plated at high density (105 cells/cm2) and were maintained for 48 h in DMEM/HAM F12 medium (Gibco BRL, Germany) supplemented with 1% penicillin/streptomycin solution (Gibco BRL, Germany), 50 mg/ml ascorbate (Sigma, Germany) and 10% fetal calf serum (Biochrom, Germany) without any change of culture medium.

900 300

the TAQMAN PCR was performed as described to detect human COL1A1, COL2A1, COL3A1, aggrecan, and MMP-1, -3, -13, -14, as well as ADAMTS-1, -4 and -5 and GAPDH [12] (Table 2). All experiments were performed in triplicates.

2.3. RNA-Isolation-cDNA synthesis-TAQMAN cDNA

2.4. cDNA array analysis-data acquisition and processing-statistical analysis

The RNA was isolated and reverse transcribed for cDNA preparation as described previously [12]. Also

The cancer 1.2 cDNA array (Clontech, Germany) was probed with 33P-dATP-labeled

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cDNA probes and the images captured and analyzed as described previously [13]. For each cell line cultured in monolayer four membranes were separately hybridized and for bead-cultures two membranes were hybridized separately. A dot was considered to be positive if it was well located, dot like, and at least 2-fold above the local background levels. Normalization of the obtained data sets was performed using the centralization method [14], which was shown to be superior to globalization approaches used in general. The significant differences in expression levels were assessed with the t-test.

3. Results The obtained expression data required a hierarchical strategy for further analysis due to the enormous amount of primary data sets. Our analytical strategy comprised three steps: (I) capturing and normalization of primary array-data. (II) categorization of interesting genes according to their cell-biological function. (III) comparison of expression levels in different culturing systems (monolayer vs. bead culture). The 15 most strongly expressed genes in the cell lines and primary articular chondrocytes are listed in Table 3. Genes with comparable expression levels in

Table 3 Table displaying the expression levels of the fifteen most strongly detected genes []: expression Intensity in arbitrary units; (): accession numbers) detected genes (together with the accession numbers) for each of the investigated cell lines (AD, SM, 105 KC) in comparison to primary human articular chondrocytes (PHC) in vitro AD

SM

105 KC

PHC

GAPDH (X01677) [2440] 40S ribosomal protein S16 (M60854) [680] 60S ribosomal protein L10 (M73791) [577] Endonuclease III homolog 1 (U79718) [450] 60S ribosomal protein L32 (X03342) [433]

GAPDH (X01677) [1724] vimentin (X56134) [563]

GAPDH (X01677) [872] Elongation factor 1 alpha (M27364) [638] Vimentin (X56134) [630]

MMP3 (X05232) [26504] Chitinase (U58514) [2726]

Ubiquitin (M26880) [445]

Fibronectin (X02761) [2066]

40S ribosomal protein S16 (M60854) [358]

Vimentin (X56134) [1100]

Fibronectin (X02761) [327]

Ubiquitin (M26880) [792]

60S ribosomal protein L32 (X03342) [304] Endonuclease III homolog 1 (U79718) [287]

Elongation factor 1 alpha (M27364) [603] 60S ribosomal protein L32 (X03342) [551]

60S ribosomal protein L13A (X56932) [287]

Decorin (M14219) [496]

60S ribosomal protein L10 (M73791) [256] TIMP3 (Z30183) [202]

Endonuclease III homolog 1 (U79718) [496] 60S ribosomal protein L13A (X56932) [455] 40S ribosomal protein S16 (M60854) [446] 60S ribosomal protein L10 (M73791) [438] Glia-derived neurite-promoting factor (A03911) [404]

Elongation factor 1 alpha (M27364) [386] Pleiotrophin (M57399) [350] Vimentin (X56134) [346]

c-myc purine-binding transcription factor puf (L16785) [315] 60S Ribosomal protein L13A (X56932) [298] 40S Ribosomal protein S5 (U14970) [235] Ubiquitin (M26880) [198] Interferon-induced 56-kDa protein (X03557) [100] Brain-specific tubulin alpha 1 subunit (K00558) [97] c-myc oncogene (V00568) [79]

40S ribosomal protein S16 (M60854) [487] 60S ribosomal protein L32 (X03342) [371] Alpha-2-macroglobulin receptor-associated protein (M63959) [334] 60S ribosomal protein L10 (M73791) [312] Elongation factor 1 alpha (M27364) [296] c-myc purine-binding transcription factor puf (L16785) [265] 60S ribosomal protein L13A (X56932) [264] Endonuclease III homolog 1 (U79718) [262] High mobility group protein (M23619) [238] Ubiquitin (M26880) [231] COL6A1 (X15879) [205] MMP14 (D26512) [187]

c-jun N-terminal kinase 2 (L31951) [163]

Cathepsin D (M11233) [168] Brain-specific tubulin alpha 1 subunit (K00558) [140] c-myc purine-binding transcription factor puf (L16785) [130] fos-related antigen (X16707) [106]

The genes are ranked by the mean of centralized expression levels in the corresponding samples.

GAPDH (X01677) [2722]

Cathepsin D (M11233) [378]

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Table 4 Table listening genes associated with cell cycle processes. The values given are normalized gene expression levels of monolayer cell lines (AD, SM, 105 KC) and primary human articular chondrocytes (PHC) Gene/protein

Acc. no.

Cell cycle function

Cyclin I

D50310

Cell cycle kinases; polymerases

Serine/threonineprotein kinase PLK1 Cell division protein kinase 4; (CDK4) Cell division protein kinase 5 (CDK5) Cyclin-dependent kinase regulatory subunit 1 (CKS1) Cyclin-dependent kinase regulatory subunit (CKS2) Proliferating cyclic nuclear antigen (PCNA); cyclin Early growth response protein 1 (hEGR1) Cyclin-dependent kinase 4 inhibitor (CDK4I; CDKN2); multiple tumor suppressor 1 (MTS1) Cyclin-dependent kinase inhibitor 1 (CDKN1A)

AD

SM

105 KC

PHC

Expr

P

Expr

P

Expr

P

Expr

nd

0,003

nd

0,003

nd

0,003

14

U01038

11

0,000

nd

ns

6

ns

0

M14505

4

ns

6

ns

16

0,001

0

X66364

5

ns

5

ns

19

0,002

0

X54941

31

0,003

15

ns

19

ns

16

X54942

22

0,000

5

ns

30

0,000

0

M15796

37

0,004

13

ns

9

ns

15

M62829

0

0,004

9

0,007

6

0,006

76

12

0,007

19

0,014

nd

ns

1

nd

0,003

8

ns

18

ns

11

L27211

U09579

CDK inhibitors

Significance (t-test) is calculated separately for each cell against primary chondrocytes. nd, not detectable; ns, not significant (PO0,05).

between cell lines and primary chondrocytes comprised the housekeeping genes including the 40S and 60S ribosomal complex as well as vimentin, a marker of the mesenchymal origin [15]. Analysis of gene expression revealed also a significant number of differentially expressed genes discussed according to their biological function in detail below. 3.1. Proliferation activity The doubling time of chondrosarcoma cell lines was between 24 and 36 h (AD 24 h, SM 36 h,

105 KC 36 h), whereas primary chondrocytes in monolayer culture had a doubling time of about 7– 10 days after passage. The increased proliferation activity of chondrosarcoma cell lines was reflected in the expression levels of genes involved in cell cycle regulation (Table 4). The serine/threonineprotein kinase PLK1, which is supposed to contribute to promotion and progression of human cancers [16] was significantly up-regulated in most cell lines. The cyclin-dependent kinase regulatory subunit 2 (CKS2) and the cell division protein kinase 5 (CDK5) were switched on only in the cell lines and

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148

Table 5 Expression of genes involved in matrix turnover as established by (a) cDNA array and (b,c) quantitative PCR analysis (a) Gene

COL1A2 Fibronectin Biglycan Tenascin Osteonectin MMP-11 MMP-13 MMP-1 MMP-2 MMP-3 MMP-14 MMP-17

Acc. no.

X55525 X02761 J04599 X78565 J03040 X57766 X75308 X05231 J03210 X05232 D26512 X89576

AD

SM

105 kc

PHC

Expr

P

Expr

P

Expr

P

Expr

nd 14 67 11 29 nd nd nd 9 nd 48 nd

ns 0,001 ns 0,085 ns 0,016 0,016 ns 0,001 0,020 ns ns

5 45 5 nd 44 nd nd nd 11 0 187 7

ns 0,001 ns 0,052 ns 0,016 0,016 ns 0,001 0,020 0,000 ns

nd 327 75 20 56 nd nd nd 56 nd 49 nd

ns 0,001 ns ns ns 0,016 0,016 ns 0,000 0,020 ns ns

35 2066 156 63 294 26 78 110 0 26504 46 4

(b)

Agg

COL1A1

COL2A1

COL2A1-A

COL10A1

CDRAP

SOX9

PHC1 PHC2 AD SM 105 KC

0,8 0,77 !0,0001 !0,0001 !0,0001

0,02 0,025 2,2 0,27 2,6

2,3 1,3 !0,0001 !0,0001 0,001

0,37 0,20 !0,0001 !0,0001 !0,0001

0,1 0,04 0,001 0,001 0,001

0,09 0,13 !0,0001 !0,0001 !0,0001

0,27 0,46 0,0005 0,003 0,0004

(c)

MMP-1

MMP-3

MMP-13

ADAMTS-1

ADAMTS-4

ADAMTS-5

PHC1 PHC2 AD SM 105 KC

1,7 3,0 0,033 0,0008 0,044

121 130 !0,0001 0,01 !0,0001

1,3 2,5 !0,0001 0,0003 !0,0001

0,29 0,23 0,11 0,12 0,16

!0,0001 !0,0001 !0,0001 0,0016 !0,0001

0,52 0,66 0,17 0,009 0,14

Significance (t-test) was calculated separately for each cell line against primary chondrocytes. nd: not detectable; ns: not significant (PO0,05). Expression levels for (b,c) real-time PCR are given as molecules/molecule GAPDH and for array analysis (a) in arbitrary units.

other kinases like the cyclin-dependent kinase regulatory subunit 1 (CKS1) were also higher expressed in most cell lines compared to primary chondrocytes. Furthermore, primary chondrocytes showed more strongly expressed genes such as EGR-1, that belongs to the class of early geneencoded transcription factors and is thought to be important in the regulation of growth factors, hormones, and cytokines [17]. 3.2. Marker genes of chondrocytic differentiation, anabolic activity, and matrix degradation In primary articular chondrocytes genes involved in matrix formation and matrix turnover like fibronectin, decorin, COL6A1, MMP-3 and TIMP-3 showed the highest expression levels, whereas they were significantly weaker expressed in

chondrosarcoma cell lines. Hardly any expression of SOX9 was detectable in the cell lines, which is known to be a marker gene of chondrocytic cell differentiation by qPCR (SOX9 was not spotted on the arrays; Table 5b). Also collagen encoding genes (COL1A2, COL2A1, COL3A1, COL6A2, COL6A3, COL11A2) could only be detected in the investigated cell lines at very low levels by the cDNA array technology as well as the more sensitive qPCR assay (Table 5). A weak signal for aggrecan could be detected in all cell lines. Fibronectin was found with a comparatively strong signal in all chondrosarcoma cells. Other noncollagenous matrix genes, like biglycan and decorin, however, were consistently switched on in all cell lines at lower expression level. Analogous to matrix formation, genes associated with matrix degradation could hardly be detected in the cell lines (Table 5c). The only exception was MMP-14, which appears to be

C.M. Scho¨rle et al. / Cancer Letters 226 (2005) 143–154

constitutively expressed by all chondrocytic cells [12,18]. Quantitative PCR (Table 5c) was able to detect low levels of MMP-1, and ADAMTS-1 -and -5 proteases. The latter were not spotted on the arrays, but are known to be expressed in articular chondrocytes in vitro and in vivo [12]. Similar to primary chondrocytes in vivo and in vitro [12], all five cell lines expressed hardly any ADAMTS-4 (Table 5c). Of note, MMP-3 was not significantly expressed in any cell line even though it is one of the most strongly expressed genes in chondrocytes in vivo and in vitro [12]. 3.3. Comparison of gene expression profiles in monolayer and alginate culture Chondrocytes are in articular cartilage embedded in a highly negatively charged extracellular matrix. The

149

interaction between chondrocytes and extracellular matrix is known to have a great impact on differentiation, growth and phenotypic properties of chondrocytes [19]. Alginate bead cultures imitating the three dimensional environment of extracellular matrix are thought to be able to preserve the chondrocyte phenotype and allow sub-cultured chondrocytes to regain a differentiated phenotype even after prolonged culture in monolayer [20,21]. Therefore, we analyzed the proliferative activity and matrix gene expression levels in alginate bead culture compared to high density monolayer culture (Table 6). Each of the cell lines cultured in monolayers showed a polygonal morphology, whereas in alginate suspension culture the cells resembled primary chondrocytes in vivo with a spherical shape (not shown). Proliferative activity of chondrosarcoma cell lines

Table 6 Table showing differentially expressed genes in monolayer and alginate culture Protein/gene

Acc. no.

AD

SM

105 KC

m

b

P

m

b

P

m

b

P

Jun-B Cyclin-dependent kinase 4 inhibitor c-jun N-terminal kinase 2 (JNK2) STAT-induced STAT inhibitor 3 rho GDP dissociation inihibitor 1 EGF response factor 1 (ERF1) Early growth response protein 1 (hEGR1) Transmembrane protein sex Integrin beta 8 Aggrecan Vascular endothelial growth factor COL6A1 TIMP1

M29039 L27211

nd 12

17 44

0,001 Ns

5 19

nd nd

ns ns

nd nd

nd 29

ns 0,008

L31951

79

48

0,041

163

569

ns

54

260

0,030

AB004904

Nd

5

Ns

5

18

ns

17

36

0,018

X69550

Nd

nd

Ns

nd

8

0,005

nd

13

0,001

X79067

26

62

Ns

24

116

0,019

27

124

0,036

M62829

Nd

10

Ns

9

81

0,001

6

114

0,024

X87852

48

25

Ns

101

327

0,023

74

100

ns

M73780 M55172 M32977

47 58 Nd

17 26 48

Ns 0,011 0,006

81 nd 22

239 4 nd

ns ns 0,005

63 12 6

83 2 26

0,032 ns ns

X15879 X03124

18 11

22 44

Ns 0,010

205 15

21 151

0,027 ns

15 103

37 116

ns ns

Significance levels were determined by t-test. m, monolayer; b, bead; nd, not detectable; ns, not significant (PO0,05).

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150

were markedly decreased in bead culture compared to monolayer cultures. This was reflected on a molecular level in a significantly increased expression of cyclindependent kinase 4 inhibitor, known to encode two distinct growth inhibitors, the cyclin-dependent kinase inhibitor p16Ink4a and the tumor suppressor p19Arf [22] in AD and 105 KC cells, respectively. In bead culture EGR-1 was significantly up-regulated in all chondrosarcoma cells, whereas no or only weak signals were detected in monolayer as discussed above. This also applied for EGF-response factor 1 (ERF-1), which is characterized by a high degree of sequence similarity with EGR-1 [23]. The anabolic synthetic activity of chondrosarcoma cells in beads, however, was not markedly increased compared to cells grown in monolayer. The aggrecan expression was only up-regulated in SM cells. No signal of COL2A1 gene expression could be detected in the cDNA array, irrespective of monolayer or bead cultures.

oncogene expression has been found [24,25]. Thus, we screened the arrays for known oncogenes and genes with oncogenic potential, relevant also in chondrosarcoma development (Table 7). C-myc, which is suggested to be involved in the development of chondrosarcoma [25], was expressed in all cell lines with a significant up-regulation in AD cells. Pleiotrophin (PTN), which has been also demonstrated as a potent promoter of angiogenesis [26], was expressed in the cell lines, whereas normal primary articular chondrocytes were negative. Of interest, pleiotrophin shows an abundant appearance in juvenile cartilage suggesting a role in chondrocyte differentiation [27]. Recently, an up-regulation of pleiotrophin has also been connected to early osteoarthritic chondrocytes with potential influence on cartilage repair [27]. Also, the MTS1 tumor suppressor gene, which is mutationally inactivated in a variety of human tumor types [28], was significantly up-regulated in neoplastic cells compared to articular chondrocytes. Decreased levels of the c-myc transcription factor puf, also known as NM23 gene, have been correlated with increased tumor metastatic potential in a variety of malignancies [29]. The NDK-A, another member of NM23 protein family, is widely documented as a suppressor of

3.4. Oncogene expression, angiogenic factors, and tumor suppressor genes So far, little is known of specific oncogenes in chondrosarcomas and no consistent pattern of

Table 7 Table showing potential oncogenes and tumor suppressor genes of chondrosarcomas known to be involved in oncogenesis of other tissues Protein/gene

Chromosome lokalisation

Acc. no.

AD

SM

105 KC

PHC

Expr

P

Expr

P

Expr

P

Expr

Pleiotrophin (PTN) c-myc oncogene Multiple tumor suppressor 1 (MTS1) Active breakpoint cluster regionrelated protein c-myc purinebinding transcription factor puf

7q33-q34

M57399

350

0,000

9

0,002

11

ns

nd

8q24.12–q24.13 9p21

V00568 L27211

79 12

0,010 0,007

14 19

ns 0,014

26 0

ns 0,5

25 1

17p13.3

U01147

24

ns

19

ns

11

ns

35

17q21.3

L16785

315

ns

265

ns

130

ns

230

X17620

nd

ns

nd

ns

nd

ns

8

Tumor suppressor genes Metastasis inhibition factor NM23

17q21.3

Significance (t-test) is calculated separately for each cell line in monolayer culture ainst primary chondrocytes (PHC). nd, not detectable; ns, not significant (PO0,05).

C.M. Scho¨rle et al. / Cancer Letters 226 (2005) 143–154

the metastatic phenotype in many human cancers. This gene was switched on only in primary chondrocytes, whereas no expression could be detected in all chondrosarcoma cell lines.

4. Discussion Adult articular chondrocytes are regarded as terminally differentiated cells that maintain their phenotype under conditions of low cellular turnover in vivo [1]. Chondrosarcoma derived cell lines might be a potential substitute promising consistent expression of a differentiated chondrocyte phenotype with sufficient proliferative activity [1,3,4]. To date, chondrosarcoma derived cell lines were predominantly analyzed for selected genes [6,7], whereas the present study presents an extensive gene profiling in correlation to primary chondrocytes. This showed only to a very limited extent a chondrocytic phenotype of the investigated chondrosarcoma cells as documented by the very low expression of SOX9, a master gene of chondrocytic differentiation [30], and cartilage typical extracellular matrix genes. Genes involved in pericellular matrix formation, like fibronectin and COL6A1 could in contrast be found in the chondrosarcoma cells. The incapability of chondrosarcoma cell lines to maintain an adequate matrix turnover as well as a notable proliferative activity at the same time is comparable to chondrosarcoma cells in vivo which cease largely cartilage matrix formation as soon as their proliferative activity increases [31]. The increased proliferation rate of chondrosarcoma cells is reflected not only in markedly accelerated doubling times compared to primary chondrocytes, but also in significantly higher expression levels of cell cycle associated proteins. In their physiological environment, chondrocytes are embedded in a dense, highly negatively charged extracellular matrix with extensive influence on growth, differentiation, and survival of chondrocytes [19]. Culturing chondrocytes in alginate is supposed to mimic these complex cell–matrix interactions and is thought to preserve the chondrocyte phenotype even in long term cultures [4,32]. Also, it allows sub-cultured chondrocytes to regain a differentiated phenotype even after prolonged culture in monolayer [20,21]. The investigated chondrosarcoma cell lines shared some of

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the basic regulation mechanisms and characteristic expression profiles of chondrocytic differentiation in a three-dimensional matrix. Thus, the proliferative activity was reduced in alginate bead culture with markedly increased doubling times of up to 5–7 days. This was also reflected by the up-regulation of kinase inhibitory proteins (i.e. cyclin-dependent kinase 4 inhibitor). Simultaneously, immediate-early genes like EGR-1, which are highly expressed in fetal human chondrocytes [33], transcriptional factors and genes involved in pericellular matrix formation (e.g. fibronectin, decorin and COL6A1) were up-regulated. This process indicates a cellular adaptation to a modified pericellular micro-environment. Recently, the chondrosarcoma cell-line SW1353 has been investigated for gene expression patterns after stimulation with interleukin-1b (IL-1b), which is known to have multiple effects on proliferate activity, activation of inflammatory responses and changes in matrix gene expresion [34,35]. The results of this study showed an early and transient response of c-Jun and JunB genes within 1 hour of IL-1 treatment [35]. The transfer of monolayer cultured neoplastic chondrocytes AG and AD to the ‘suspension’ alginate bead system in our study revealed a similar increase of gene expression levels with a strong up-regulation of c-Jun, EGR-1 and JunB. The responsiveness and activation of immediate-early genes in both conditions clearly indicates that chondrocytes show not only a distinct change in gene expression patterns after exposure to specific mediators and cytokines, but also a similar reaction to certain culture conditions such as threedimensional matrices. The effect of the surrounding extracellular matrix on the expression patterns in chondrocytic cells is also reflected by the regulation of c-jun N-terminal kinase 2 (JNK2), a member of the MAP kinases, which mediate signals from the cell surface to the nucleus. Again, the increased levels of JNK2 expression, found in bead culture and induced by IL-1b further confirms that signal transduction pathways thought to mediate mostly cytokine signaling are also reactive to changes in cell–matrix interactions. Interestingly, besides EGR-1 also casein kinase II showed an increased expression level in bead culture of AG and AD cells maybe as a compensatory mechanism: this kinase is known to be active on EGR-1, thereby preventing DNA-binding and gene activation [36] by this molecule.

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The chondrocyte-typical synthetic activity of chondrosarcoma cells was only slightly increased in alginate beads, demonstrated only in a sporadic upregulation of aggrecan and still a very low expression of COL2A1. Of note, the aggrecan expression of another chondrosarcoma-derived cell line HCS-2/8 increased with exposure to hydrostatic pressure, imitating the natural environment of cartilage tissue in which chondrocytes are constantly influenced by mechanical stress [37]. The rate of aggrecan synthesis in this study was increased with exposure to low, intermittent pressure and decreased under static pressure [38], suggesting that specific mechanical loading protocols may be required to optimally promote aggrecan synthesis [37]. Thus, further studies will have to elucidate whether similar loading protocols will be able to induce aggrecan expression also in the cell lines investigated in this study. However, HCS-2/8 cells show strikingly higher levels of aggrecan expression also without stimulation [39]. Altogether, the chondrosarcoma - derived cell lines analyzed in this study - at least under the culture conditions tested - showed only to a limited extent a chondrocytic phenotype. This does not exclude their potential usefulness as in vitro model systems for studying chondrocyte behavior, but clearly points out that any result of such studies needs to be carefully reconfirmed in primary chondrocytes or even better cartilage-tissue cultures. This was recently demonstrated for IL-1 stimulation of other chondrosarcoma cell lines SW1353 ([40]; own unpublished results) as well as OUMS-27 [41], of which at least also SW1353 cells express markers of chondrocytic differentiation only at a very low level (own unpublished results). The limited resemblance of chondrosarcoma derived cells with primary chondrocytes to physiological chondrocytes is most likely related to their neoplastic origin and, thus, these cells might have a much higher potential to investigate the behavior of neoplastic chondrocytes, i.e. chondrosarcoma biology. In the present study, the chondrosarcoma cell lines were all positive for c-myc and expression levels were significantly increased in some cell lines compared to primary chondrocytes. The scarcity of altered expression levels of further oncogenes is in line with a previous study detecting c-myc as the sole proto-oncogene amplified in chondrosarcomas, whereas no other oncogenes studied were amplified

[25]. Tumor invasion potential is mainly correlated with tumor neovascularization. Pleiotrophin (PTN) which has been demonstrated as potent promoter of angiogenesis [26], was expressed in all cell lines, whereas primary articular chondrocytes were negative. This is in line with the notion that chondrosarcoma cells can induce angioinvasive factors and correlates well with the fact that all cell lines were derived from high grade chondrosarcomas, lesions known to show rather high vascular density in contrast to physiological cartilage and benign or low grade chondroid tumors. In our study, we used gene expression profiling by the array-technology as well as quantitative PCR. Recently, another approach for analyzing gene expression under specific conditions has been presented with the systematical exploration of cDNA libraries [42]. These procedure using random sequencing of large numbers of expressed sequence tags (ESTs) enables gene expression profiling and - in theory - the identification of novel genes [42]. The most highly expressed genes of human chondrosarcoma cell-line HCS-2/8 found with this strategy comprised fibronectin, fibromudulin, decorin, growth factors like connective tissue growth factor and the common housekeeping genes [42]. This is in clear correspondence with the 15 most strongly detected genes in the cell lines AG, AD and 105 KC. In conclusion, the investigated chondrosarcoma derived cell lines do show significant alterations in their gene expression profile compared to isolated primary human articular chondrocytes reminiscent to features typical for neoplastic chondrocytes in vivo. Thus, they might be most of all helpful in studying the behavior of neoplastic chondrocytes in vitro. This is in particular relevant, as no biological therapeutic approaches are so far available for the treatment of chondrogenic neoplasms and surgery is so far the only curative option in cases of localized disease with basically hardly any option left in metastatic disease.

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